Synthetic molecule constructs

ABSTRACT

Synthetic molecule construct of the structure F-S 1 -S 2 -L where F-S 1  is an aminoalkylglycoside and S 2 -L is a dicarboxylated, e.g. adipylated, phosphatidylethanolamide are disclosed. The two terminal sugars of the aminoalkylglycoside (Galα1-3Galβ-) are the two terminal sugars of the Galili antigen.

This application is a continuation-in-part (CIP) of U.S. applicationSer. No. 15/804,427 filed Nov. 6, 2017, which is a CIP of Ser. No.15/168,144 filed May 30, 2016 (U.S. Pat. No. 9,809,614), which is acontinuation of Ser. No. 14/108,749 filed Dec. 17, 2013 (U.S. Pat. No.9,353,349), which is a continuation of Ser. No. 13/067,021 filed May 3,2011 (U.S. Pat. No. 8,637,473), which is a divisional of Ser. No.10/593,829 filed Jan. 12, 2007 (U.S. Pat. No. 8,013,131), which is theU.S. national phase of PCT/NZ2005/000052 filed Mar. 22, 2005, whichclaims priority to New Zealand Patent Application Nos. 531866 filed Mar.22, 2004, and 537941 filed Jan. 28, 2005, the contents of each of whichare hereby incorporated by reference. Ser. No. 15/804,427 is also a CIPof Ser. No. 14/972,301 filed Dec. 17, 2015 which is a CIP of Ser. No.14/085,156 filed Nov. 20, 2013 (U.S. Pat. No. 9,226,968), which is acontinuation of Ser. No. 13/459,399 filed Apr. 30, 2012 (abandoned),which is a continuation of Ser. No. 12/451,120 filed Mar. 29, 2010 (U.S.Pat. No. 8,211,860), which is the U.S. national phase ofPCT/NZ2008/000095 filed Apr. 28, 2008, the contents of each of which arehereby incorporated by reference.

This application is also a CIP of Ser. No. 15/279,652 filed Sep. 29,2016, which is a continuation of Ser. No. 14/563,127 filed Dec. 8, 2014,which is a continuation of Ser. No. 13/354,449 filed Jan. 20, 2012(abandoned), which is a continuation of Ser. No. 12/310,803 filed Mar.4, 2010 (abandoned), which is the U.S. national phase ofPCT/NZ2007/000256 filed Sep. 6, 2007, which claims priority to NewZealand Patent Application Nos. 549742 filed Sep. 6, 2006, and 549740filed Sep. 7, 2006, the contents of each of which are herebyincorporated by reference.

FIELD OF INVENTION

The invention relates to synthetic molecules that spontaneously andstably incorporate into lipid bilayers, including cell membranes.Particularly, although not exclusively, the invention relates to the useof these constructs to effect qualitative and quantitative changes inthe expression of antigens and flurorophores at the surface of cells.

BACKGROUND

Cell surface antigens mediate a range of interactions between cells andtheir environment. These interactions include cell-cell interactions,cell-surface interactions and cell-solute interactions. Cell surfaceantigens also mediate intra-cellular signalling.

Cells are characterised by qualitative and quantitative differences inthe cell surface antigens expressed. Qualitative and quantitativechanges in the cell surface antigens expressed alter both cell function(mode of action) and cell functionality (action served).

The marking of cells by conjugation of a fluorophore with a surfaceexpressed antigen may affect cell function. Furthermore, mobility of thefluorophore within the two dimensions of the cell membrane isnecessarily dependent on the mobility of the conjugated antigen.

Being able to localise fluorophores to the surface of cells withoutthese limitations or effect qualitative and/or quantitative changes inthe surface antigens expressed by a cell has diagnostic and therapeuticvalue. Transgenic and non-transgenic methods of effecting qualitativeand/or quantitative changes in the surface antigens expressed by a cellare known.

Protein painting is a non-transgenic method for effecting qualitativeand/or quantitative changes in the surface antigens expressed by a cell.The method exploits the ability of GPI linked proteins to spontaneouslyanchor to the cell membrane via their lipid tails. The method describedin the specification accompanying international application no.PCT/US98/15124 (publ. no. WO 99/05255) includes the step of inserting aGPI linked protein isolated from a biological source into a membrane.Isolated GPI-anchored proteins are stated as having an unusual capacityto reintegrate with a cell-surface membrane.

Cells exist in an aqueous environment. The cell membrane is a lipidbilayer that serves as a semi-permeable barrier between the cytoplasm ofthe cell and this aqueous environment. Localising antigens to the cellsurface may also be achieved by the use of glycolipids as membraneanchors.

The method described in the specification accompanying internationalapplication no. PCT/NZ02/00214 (publ. no. WO 03/034074) includes thestep of inserting a controlled amount of glycolipid into a membrane. Theamount of glycolipid inserted is controlled to provide cells with adesired level of antigen expression.

The method described in the specification accompanying internationalapplication no. PCT/NZ03/00059 (publ. no. WO 03/087346) includes thestep of inserting a modified glycolipid into a membrane as a “membraneanchor”. The modified glycolipid provides for the localisation ofantigens to the surface of the cell or multicellular structure. Newcharacteristics may thereby be imparted on the cell or multicellularstructure.

These methods typically include the isolation of a glycolipid orglycolipid-linked antigen from a biological source. The isolation ofglycolipids or glycolipid-linked antigens from biological sources iscostly, variable and isolatable amounts are often limited. Obtainingreagents from zoological sources for therapeutic use is particularlyproblematic, especially where the reagent or its derivative products areto be administered to a human subject.

Synthetic molecules for which the risk of contamination withzoo-pathogenic agents can be excluded are preferred. Syntheticcounterparts for naturally occurring glycolipids and syntheticneo-glycolipids have been reported. However, for a synthetic glycolipidto be of use as a membrane anchor it must be able to spontaneously andstably incorporate into a lipid bi-layer from an aqueous environment.The utility of synthetic glycolipids in diagnostic or therapeuticapplications is further limited to those synthetic glycolipids that willform a solution in saline.

Organic solvents and/or detergents used to facilitate the solubilizationof glycolipids in saline must be biocompatible. Solvents and detergentsmust often be excluded or quickly removed as they can be damaging tosome cell membranes. The removal of solvents or detergents from suchpreparations can be problematic.

Damage to cell membranes is to be avoided especially where the supply ofcells or multicellular structures is limited, e.g. embryos, or the cellsare particularly sensitive to perturbation, e.g. hepatocytes.

Methods of localising fluorophores and antigen to the surface of cellsthat avoid affecting cell function and provide for independent mobilityof the fluorophore or antugen within the two dimensions of the cellmembrane are desired.

There exists a need for water soluble synthetic molecules that can beused to localise fluorophores to the surface of cells or arefunctionally equivalent to naturally occurring glycolipids andglycolipid-linked antigens in respect of their ability to spontaneouslyand stably incorporate into lipid bilayers, including cell membranes.

Providing such synthetic molecules would obviate the limitations ofmarking cells by conjugation of a fluorophore with a surface expressedantigen. Providing such synthetic molecules would also obviate thelimitations of glycolipids and glycolipid-linked antigens isolated frombiological sources and facilitate being able to effect qualitativeand/or quantitative changes in the surface antigens expressed by a cell.

It is an object of this invention to provide such synthetic moleculesand a method for their preparation. It is a further object of thisinvention to provide synthetic molecules for use in diagnostic andtherapeutic applications. The preceding objects are to be readdisjunctively with the object to at least provide the public with auseful choice.

STATEMENTS OF INVENTION

In a first aspect the invention provides a synthetic molecule constructof the structure F-S₁-S₂-L where:

-   -   F is selected from the group consisting of carbohydrates and        fluorophores;    -   S₁-S₂ is a spacer linking F to L; and    -   L is a lipid selected from the group consisting of diacyl- and        dialkyl-glycerolipids, including glycerophospholipids, and        sphingosine derived diacyl- and dialkyl-lipids, including        ceramide.

Preferably, L is a lipid selected from the group consisting of diacyl-and dialkyl-glycerolipids, including glycerophospholipids. Morepreferably, L is selected from the group consisting of:diacylglycerolipids, phosphatidate, phosphatidyl choline, phosphatidylethanolamine, phosphatidyl serine, phosphatidyl inositol, phosphatidylglycerol, and diphosphatidyl glycerol derived from one or more oftrans-3-hexadecenoic acid, cis-5-hexadecenoic acid, cis-7-hexadecenoicacid, cis-9-hexadecenoic acid, cis-6-octadecenoic acid,cis-9-octadecenoic acid, trans-9-octadecenoic acid,trans-11-octadecenoic acid, cis-11-octadecenoic acid, cis-1-eicosenoicacid or cis-13-docsenoic acid. More preferably, the lipid is derivedfrom one or more cis-destaurated fatty acids. Most preferably, L isselected from the group consisting of:1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE),1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE) andrac-1,2-dioleoylglycerol (DOG).

Preferably, L is a glycerophospholipid and the molecule includes thesubstructure:

where n is 3 to 5, X is H or C, and

is other than H. Preferably, n is 3. Preferably, the molecule is watersoluble.

Preferably, the molecule spontaneously incorporates into a lipidbi-layer when a solution of the molecule is contacted with the lipidbi-layer. More preferably, the molecule stably incorporates into thelipid bilayer.

Preferably, F, S₁, S₂ and L are covalently linked.

S₁-S₂ is selected to provide a water-soluble synthetic moleculeconstruct.

In a first embodiment F is a naturally occurring or synthetic glycotope.Preferably, F is a naturally occurring or synthetic glycotope consistingof three (trisaccharide) or more sugar units. More preferably, F is aglycotope selected from the group consisting of lacto-neo-tetraosyl,lactotetraosyl, lacto-nor-hexaosyl, lacto-iso-octaosyl, globoteraosyl,globo-neo-tetraosyl, globopentaosyl, gangliotetraosyl, gangliotriaosyl,gangliopentaosyl, isoglobotriaosyl, isoglobotetraosyl, mucotriaosyl andmucotetraosyl series of oligosaccharides. Most preferably, F is selectedfrom the group of glycotopes comprising the terminal sugars:GalNAcα1-3(Fucα1-2)Galβ; Galα1-3Galβ; Galβ; Galα1-3(Fucα1-2)Galβ;NeuAcα2-3Galβ; NeuAcα2-6Galβ; Fucα1-2Galβ;Galβ1-4GlcNAcβ1-6(Galβ1-4GlcNAcβ1-3)Galβ;Fucα1-2Galβ1-4GlcNAcβ1-6(Fucα1-2Galβ1-4GlcNAcβ1-3)Galβ;Fucα1-2Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-4GlcNAcβ1-3)Galβ;NeuAcα2-3Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-4GlcNAcβ1-3)Galβ;Galα1-4Galβ1-4Glc; GalNAcβ1-3Galα1-4Galβ1-4Glc;GalNAcα1-3GalNAcβ1-3Galα1-4Galβ1-4Glc; orGalNAcβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glc.

When F is a glycotope, L is a glycerophospholipid and S₂ is selectedfrom the group including: —CO(CH₂)₃CO—, —CO(CH₂)₄CO— (adipate),—CO(CH₂)₅CO— and —CO(CH₂)₅NHCO(CH₂)₅CO—, preferably S₁ is aC₃₋₅-aminoalkyl selected from the group consisting of: 3-aminopropyl,4-aminobutyl, or 5-aminopentyl. More preferably S₁ is 3-aminopropyl.

In a second embodiment F is a molecule that mediates a cell-cell orcell-surface interaction. Preferably F is a carbohydrate with anaffinity for a component expressed on a targeted cell or surface. Morepreferably F has an affinity for a component expressed on epithelialcells or extra-cellular matrices. Yet more preferably F has an affinityfor a component expressed on the epithelial cells or the extra-cellularmatrix of the endometrium. Most preferably the component expressed onthe epithelial cells or the extra-cellular matrix of the endometrium canbe a naturally expressed component or an exogenously incorporatedcomponent.

In a third embodiment F is a molecule that mediates a cell-soluteinteraction. Preferably F is a ligand for a binding molecule where thepresence of the binding molecule is diagnostic for a pathologicalcondition. More preferably F is a ligand for an antibody(immunoglobulin).

In a fourth embodiment F is a fluorophore. Preferably, F is afluorophore selected from the group consisting of: fluorophores offluorescein, Oregon Green, Pennsylvania Green, Tokyo Green, eosin,BODIPY, BODIPY TR, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488,Alexa Fluor 568, Alexa Fluor 594, Texas Red, Lucifer Yellow,tetramethylrhodamine and their derivatives. Most preferably, F isselected from the group consisting of: fluorophores of fluorescein,BODIPY and their derivatives.

Preferably, where F is the fluorophore of fluorescein or one of itsderivatives, S₁ is a C₃₋₅-diaminoalkyl derivative selected from thegroup consisting of: 1,3-diaminopropyl, 1,4-diaminobutyl, or1,5-aminopentyl derivatives. More preferably, where F is the fluorophoreof fluorescein or one of its derivatives, S₁ is aC₃₋₅-aminoalkylthioureidyl. Most preferably, where F is the fluorophoreof fluorescein or one of its derivatives, S₁ is 5-((5-aminopentyl)thioureidyl.

Preferably, where F is the fluorophore of fluorescein or one of itsderivatives, S₂ is selected from the group including: —CO(CH₂)₃CO—, —CO(CH₂)₄CO—, —CO(CH₂)₅CO— and —CO(CH₂)₅NHCO(CH₂)₅CO—. More preferably,where F is the fluorophore of fluorescein or one of its derivatives, S₂is —CO(CH₂)₄CO—.

Preferably, where F is the fluorophore of fluorescein or one of itsderivatives, the structure includes the substructure:

where m and n are independently 3 to 5 and

is other than H.

Preferably, where F is the fluorophore of BODIPY or one of itsderivatives, S₁ is a C₃₋₅-alkionyldiamine. More preferably, where F isthe fluorophore of BODIPY or one of its derivatives, S₁ is propionylethyldiamine.

Preferably, where F is the fluorophore of BODIPY or one of itsderivatives, S₂ is selected from the group consisting of: —CO(CH₂)₃CO—,—CO(CH₂)₄CO— and —CO(CH₂)₅CO—. More preferably, where F is thefluorophore of BODIPY or one of its derivatives, S₂ is —CO(CH₂)₄CO—.

Preferably, where F is the fluorophore of BODIPY or one of itsderivatives the structure includes the substructure:

where p, q and r are independently 3 to 5 and * is other than H. Morepreferably, the sum of p, q and r is B. Most preferably, p is 2, q is 2and r is 4.

In specific embodiments the synthetic molecule construct has thestructure:

the structure:

the structure:

the structure:

the structure:

the structure:

the structure:

the structure:

the structure:

the structure:

the structure:

the structure:

the structure:

the structure:

or the structure:

M is typically H, but may be replaced by another monovalent cation suchas Na⁺, K⁺ or NH₄ ⁺.

In a second aspect the invention provides a method of preparing asynthetic molecule construct of the structure F-S₁-S₂-L including thesteps:

-   1. Reacting an activator (A) with a lipid (L) to provide an    activated lipid (A-L);-   2. Derivatising an antigen (F) to provide a derivatised antigen    (F-S₁); and-   3. Condensing A-L with F-S₁ to provide the molecule;-   where:    -   A is an activator selected from the group including:        bis(N-hydroxysuccinimidyl), bis(4-nitrophenyl),        bis(pentafluorophenyl), bis(pentachlorophenyl) esters of        carbodioic acids (C₃ to C₇);    -   L is a lipid selected from the group consisting of diacyl- and        dialkyl-glycerolipids, including glycerophospholipids, and        sphingosine derived diacyl- and dialkyl-lipids, including        ceramide.    -   F is selected from the group consisting of carbohydrates; and    -   S₁—S₂ is a spacer linking F to L where S₁ is selected from the        group including: primary aminoalkyl, secondary aliphatic        aminoalkyl or primary aromatic amine; and S₂ is absent or        selected from the group including: —CO(CH₂)₃CO—, —CO(CH₂)₄CO—        (adipate), and —CO(CH₂)₅CO—.

Preferably, the molecule is water soluble.

Preferably, the molecule spontaneously incorporates into a lipidbi-layer when a solution of the molecule is contacted with the lipidbi-layer. More preferably the molecule stably incorporates into thelipid bilayer.

Preferably, F, S₁, S₂ and L are covalently linked.

Preferably, F is selected from the group consisting of naturallyoccurring or synthetic glycotopes.

Preferably, L is a lipid selected from the group consisting of diacyl-and dialkyl-glycerolipids, including glycerophospholipids. Morepreferably L is selected from the group consisting of:diacylglycerolipids, phosphatidate, phosphatidyl choline, phosphatidylethanolamine, phosphatidyl serine, phosphatidyl inositol, phosphatidylglycerol, and diphosphatidyl glycerol derived from one or more oftrans-3-hexadecenoic acid, cis-5-hexadecenoic acid, cis-7-hexadecenoicacid, cis-9-hexadecenoic acid, cis-6-octadecenoic acid,cis-9-octadecenoic acid, trans-9-octadecenoic acid,trans-1l-octadecenoic acid, cis-11-octadecenoic acid, cis-11-eicosenoicacid or cis-13-docsenoic acid. More preferably the lipid is derived fromone or more cis-destaurated fatty acids. Most preferably L is selectedfrom the group consisting of:1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE),1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE) andrac-1,2-dioleoylglycerol (DOG).

Preferably, L is a glycerophospholipid and the molecule includes thesubstructure:

where n=3 to 5, X is H or C, and

is other than H. Preferably n is 3.

Preferably, A (R-S₂) and S₁ are selected to provide a water solublesynthetic molecule construct.

In a first embodiment F is a naturally occurring or synthetic glycotope.

Preferably, F is a naturally occurring or synthetic glycotope consistingof three (trisaccharide) or more sugar units. More preferably, F is aglycotope selected from the group consisting of lacto-neo-tetraosyl,lactotetraosyl, lacto-nor-hexaosyl, lacto-iso-octaosyl, globoteraosyl,globo-neo-tetraosyl, globopentaosyl, gangliotetraosyl, gangliotriaosyl,gangliopentaosyl, isoglobotriaosyl, isoglobotetraosyl, mucotriaosyl andmucotetraosyl series of oligosaccharides. Most preferably, F is selectedfrom the group of glycotopes comprising the terminal sugarsGalNAcα1-3(Fucα1-2)Galβ; Galα1-3Galβ; Galβ; Galα1-3(Fucα1-2)Galβ;NeuAcα2-3Galβ; NeuAcα2-6Galβ; Fucα1-2Galβ;Galβ1-4GlcNAcβ1-6(Galβ1-4GlcNAcβ1-3)Galβ;Fucα1-2Galβ1-4GlcNAcβ1-6(Fucα1-2Galβ1-4GlcNAcβ1-3)Galβ;Fucα1-2Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-4GlcNAcβ1-3)Galβ;NeuAcα2-3Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-4GlcNAcβ1-3)Galβ;Galα1-4Galβ1-4Glc; GalNAcβ1-3Galα1-4Galβ1-4Glc;GalNAcα1-3GalNAcβ1-3Galα1-4Galβ1-4Glc; orGalNAcβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glc.

When F is a glycotope, L is a glycerophospholipid and S₂ is selectedfrom the group including: —CO(CH₂)₃CO—, —CO(CH₂)₄CO—, —CO(CH₂)₅CO— and—CO(CH₂)₅NHCO(CH₂)₅CO—. Preferably, S₁ is a C₃₋₅-aminoalkyl selectedfrom the group consisting of: 3-aminopropyl, 4-aminobutyl, or5-aminopentyl. More preferably, S₁ is 3-aminopropyl.

In a second embodiment F is a molecule that mediates a cell-cell orcell-surface interaction. Preferably, F is carbohydrate with an affinityfor a component expressed on a targeted cell or surface. Morepreferably, F has an affinity for a component expressed on epithelialcells or extra-cellular matrices. Yet more preferably, F has an affinityfor a component expressed on the epithelial cells or the extra-cellularmatrix of the endometrium. Most preferably, the component expressed onthe epithelial cells or the extra-cellular matrix of the endometrium canbe a naturally expressed component or an exogenously incorporatedcomponent.

In a third embodiment F is a molecule that mediates a cell-soluteinteraction. Preferably F is a ligand for a binding molecule where thepresence of the binding molecule is diagnostic for a pathologicalcondition. More preferably F is a ligand for an antibody(immunoglobulin).

In specific embodiments the water-soluble synthetic molecule constructhas the structure:

the structure:

the structure:

the structure:

the structure:

the structure:

the structure:

the structure:

or the structure:

M is typically H, but may be replaced by another monovalent cation suchas Na⁺, K⁺ or NH₄ ⁺.

In a third aspect the invention provides a water-soluble syntheticmolecule construct prepared by a method according to the second aspectof the invention.

In a fourth aspect the invention provides a method of effectingqualitative and/or quantitative changes in the surface antigensexpressed by a cell or multi-cellular structure including the step ofcontacting a suspension of the cell or multi-cellular structure with asynthetic molecule construct according to the first aspect or thirdaspect of the invention for a time and at a temperature sufficient toeffect the qualitative and/or quantitative change in the surfaceantigens expressed by the cell or multi-cellular structure.

Preferably, the cell or multi-cellular structure is of human or murineorigin.

Preferably, the concentration of the water-soluble synthetic membraneanchor or synthetic molecule construct in the suspension is in the range0.1 to 10 mg/mL.

Preferably, the temperature is in the range 2 to 37° C. More preferablythe temperature is in the range 2 to 25° C. Most preferably, thetemperature is in the range 2 to 4° C.

In a first embodiment the cell is a red blood cell.

In this embodiment, F is preferably selected from the group ofglycotopes comprising the terminal sugars GalNAcα1-3(Fucα1-2)Galβ;Galα1-3Galβ; Galβ; Galα1-3(Fucα1-2)Galβ; NeuAcα2-3Galβ; NeuAcα2-6Galβ;Fucα1-2Galβ; Galβ1-4GlcNAcβ1-6(Galβ1-4GlcNAcβ1-3)Galβ;Fucα1-2Galβ1-4GlcNAcβ1-6(Fucα1-2Galβ1-4GlcNAcβ1-3)Galβ;Fucα1-2Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-4GlcNAcβ1-3)Galβ;NeuAcα2-3Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-4GlcNAcβ1-3)Galβ;Galα1-4Galβ1-4Glc; GalNAcβ1-3Galα1-4Galβ1-4Glc;GalNAcα1-3GalNAcβ1-3Galα1-4Galβ1-4Glc; orGalNAcβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glc. More preferably, F is selectedfrom the group of glycotopes consisting of the oligosaccharidesGalNAcα1-3(Fucα1-2)Galβ and Galα1-3(Fucα1-2)Galβ.

Preferably, the synthetic molecule construct is selected from the groupincluding: A_(tri)-sp-Ad-DOPE (I); A_(tri)-spsp₁-Ad-DOPE (II);A_(tri)-sp-Ad-DSPE (III); B_(tri)-sp-Ad-DOPE (VI); H_(tri)-sp-Ad-DOPE(VII); H_(di)-sp-Ad-DOPE (VIII); Galβ_(i)-sp-Ad-DOPE (IX);Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc-sp-Ad-DOPE (XII); andFucα1-2Galβ1-3(Fucα1-4)GlcNAc-sp-Ad-DOPE (XIII).

In a second embodiment the multi-cellular structure is an embryo.

In this embodiment, F preferably is an attachment molecule where theattachment molecule has an affinity for a component expressed on theepithelial cells or the extra-cellular matrix of the endometrium.

The component expressed on the epithelial cells or the extra-cellularmatrix of the endometrium can be a naturally expressed component or anexogenously incorporated component.

Preferably, the synthetic membrane anchor or synthetic moleculeconstruct is selected from the group including: A_(tri)-sp-Ad-DOPE (I);A_(tri)-spsp₁-Ad-DOPE (II); A_(tri)-sp-Ad-DSPE (III); B_(tri)-sp-Ad-DOPE(VI); H_(tri)-sp-Ad-DOPE (VII); H_(di)-sp-Ad-DOPE (VIII);Galβ_(i)-sp-Ad-DOPE (IX);Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc-sp-Ad-DOPE (XII); andFucα1-2Galβ1-3(Fucα1-4)GlcNAc-sp-Ad-DOPE (XIII).

In a third embodiment the cell is red blood cell.

In this embodiment, F is preferably a ligand for a binding moleculewhere the presence of the binding molecule is diagnostic for apathological condition. More preferably, F is a ligand for an antibody(immunoglobulin).

In a fifth aspect the invention consists in a cell or multi-cellularstructure incorporating a water-soluble synthetic molecule constructaccording to the first or third aspect of the invention.

Preferably, the cell or multi-cellular structure is of human or murineorigin.

In a first embodiment the cell is a red blood cell incorporating a watersoluble synthetic molecule construct selected from the group including:A_(tri)-sp-Ad-DOPE (I); A_(tri)-spsp₁-Ad-DOPE (II); A_(tri)-sp-Ad-DSPE(III); B_(tri)-sp-Ad-DOPE (VI); H_(tri)-sp-Ad-DOPE (VII);H_(di)-sp-Ad-DOPE (VIII); Galβ_(i)-sp-Ad-DOPE (IX);Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc-sp-Ad-DOPE (XII); andFucα1-2Galβ1-3(Fucα1-4)GlcNAc-sp-Ad-DOPE (XIII).

In a second embodiment the multi-cellular structure is an embryoincorporating a water soluble synthetic molecule construct selected fromthe group consisting of: A_(tri)-sp-Ad-DOPE (I); A_(tri)-spsp₁-Ad-DOPE(II); A_(tri)-sp-Ad-DSPE (III); B_(tri)-sp-Ad-DOPE (VI);H_(tri)-sp-Ad-DOPE (VII); H_(di)-sp-Ad-DOPE (VIII); Galβ_(i)-sp-Ad-DOPE(IX); Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc-sp-Ad-DOPE (XII); andFucα1-2Galβ1-3(Fucα1-4)GlcNAc-sp-Ad-DOPE (XIII).

In a sixth aspect the invention consists in a kit comprising a driedpreparation or solution of a water-soluble synthetic membrane anchor orsynthetic molecule construct according to the first or third aspect ofthe invention.

Preferably, the water soluble synthetic molecule construct according tothe first or third aspect of the invention is selected from the groupconsisting of: A_(tri)-sp-Ad-DOPE (I); A_(tri)-spsp₁-Ad-DOPE (II);A_(tri)-sp-Ad-DSPE (III); B_(tri)-sp-Ad-DOPE (VI); H_(tri)-sp-Ad-DOPE(VII); H_(di)-sp-Ad-DOPE (VIII); Galβ_(i)-sp-Ad-DOPE (IX);Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc-sp-Ad-DOPE (XII); andFucα1-2Galβ1-3(Fucα1-4)GlcNAc-sp-Ad-DOPE (XIII).

In a seventh aspect the invention consists in a kit comprising asuspension in a suspending solution of cells or multi-cellularstructures according to the fifth aspect of the invention.

Preferably, the suspending solution is substantially free of lipid.

Preferably, the cell or multi-cellular structure is of human or murineorigin.

Preferably, the cells are red blood cells that do not naturally expressA- or B-antigen and incorporate a water soluble synthetic moleculeconstruct selected from the group consisting of: A_(tri)i-sp-Ad-DOPE(I); A_(tri)-spsp₁-Ad-DOPE (II); A_(tri)-sp-Ad-DSPE (III);B_(tri)-sp-Ad-DOPE (VI); H_(tri)-sp-Ad-DOPE (VII); H_(di)-sp-Ad-DOPE(VIII); Galβ_(i)-sp-Ad-DOPE (IX);Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc-sp-Ad-DOPE (XII); andFucα1-2Galβ1-3(Fucα1-4)GlcNAc-sp-Ad-DOPE (XIII). More preferably, thecells are sensitivity controls.

In an eighth aspect the invention consists in a pharmaceuticalpreparation comprising a dried preparation or solution of awater-soluble synthetic molecule construct according to the first orfourth aspect of the invention.

Preferably, the pharmaceutical preparation is in a form foradministration by inhalation.

Preferably, the pharmaceutical preparation is in a form foradministration by injection.

In a ninth aspect the invention consists in a pharmaceutical preparationcomprising cells or multi-cellular structures according to the fifthaspect of the invention.

Preferably, the cells or multi-cellular structures are of human ormurine origin.

Preferably, the pharmaceutical preparation is in a form foradministration by inhalation.

Preferably, the pharmaceutical preparation is in a form foradministration by injection.

In a tenth aspect the invention provides a fluorescent cell marker ofthe structure:

-   -   F-S₁-S₂-L        including the substructure:

where

-   -   F is a fluorophore;    -   S₁—S₂ is a spacer linking F to L;    -   L is a lipid selected from the group consisting of diacyl- and        dialkyl-glycerolipids, including glycerophospholipids;    -   m and n are independently 3 to 6;    -   R₁ is O or S; and

is other than H.

The spacer (S₁—S₂) is selected to provide a water-soluble cell marker.

Preferably, F is selected from the group consisting of: fluorophores offluorescein, Oregon Green, Pennsylvania Green, Tokyo Green, eosin,BODIPY, BODIPY TR, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488,Alexa Fluor 568, Alexa Fluor 594, Texas Red, Lucifer Yellow,tetramethylrhodamine and their derivatives. Most preferably, F isselected from the group consisting of: fluorophores of fluorescein,BODIPY and their derivatives.

Preferably, the sum of m and n is 6 to 9 and * is C or N.

Preferably, where F is the fluorophore of fluorescein or one of itsderivatives, S₁ is a C₃₋₅-diaminoalkyl derivative selected from thegroup consisting of: 1,3-diaminopropyl, 1,4-diaminobutyl, or1,5-aminopentyl derivatives. More preferably, where F is the fluorophoreof fluorescein or one of its derivatives, S₁ is aC₃₋₅-aminoalkylthioureidyl. Most preferably, where F is the fluorophoreof fluorescein or one of its derivatives, S₁ is 5-((5-aminopentyl)thioureidyl.

Preferably, where F is the fluorophore of fluorescein or one of itsderivatives, S₂ is selected from the group including: —CO(CH₂)₃CO—,—CO(CH₂)₄CO— (adipate), —CO(CH₂)₅CO— and —CO(CH₂)₅NHCO(CH₂)₅CO—. Morepreferably, where F is the fluorophore of fluorescein or one of itsderivatives, S₂ is —CO(CH₂)₄CO— (adipate).

Preferably, where F is the fluorophore of fluorescein or one of itsderivatives, the structure includes the substructure:

where m and n are independently 3 to 5 and is other than H.

is other than H.Preferably, where F is the fluorophore of BODIPY or one of itsderivatives, S₁ is a C₃₋₅-alkionyldiamine. More preferably, where F isthe fluorophore of BODIPY or one of its derivatives, S₁ is propionylethyldiamine.

Preferably, where F is the fluorophore of BODIPY or one of itsderivatives, S₂ is selected from the group consisting of: —CO(CH₂)₃CO—,—CO(CH₂)₄CO— (adipate) and —CO(CH₂)₅CO—. More preferably, where F is thefluorophore of BODIPY or one of its derivatives, S₂ is —CO(CH₂)₄CO—(adipate).

Preferably, where F is the fluorophore of BODIPY or one of itsderivatives the structure includes the substructure:

where p, q and r are independently 3 to 5 and

is other than H. More preferably, the sum of p, q and r is 8. Mostpreferably, p is 2, q is 2 and r is 4.

Preferably L is a lipid selected from the group consisting of diacyl-and dialkyl-glycerolipids, including glycerophospholipids. Morepreferably L is selected from the group consisting of:diacylglycerolipids, phosphatidate, phosphatidyl choline, phosphatidylethanolamine, phosphatidyl serine, phosphatidyl inositol, phosphatidylglycerol, and diphosphatidyl glycerol derived from one or more oftrans-3-hexadecenoic acid, cis-5-hexadecenoic acid, cis-7-hexadecenoicacid, cis-9-hexadecenoic acid, cis-6-octadecenoic acid,cis-9-octadecenoic acid, trans-9-octadecenoic acid,trans-11-octadecenoic acid, cis-11-octadecenoic acid, cis-11-eicosenoicacid or cis-13-docsenoic acid. More preferably the lipid is derived fromone or more cis-desaturated fatty acids. Most preferably L is selectedfrom the group consisting of:1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE),1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE) andrac-1,2-dioleoylglycerol (DOG).

In a eleventh aspect the invention provides a method of marking cellsincluding the step of contacting a suspension of cells with a cellmarker of the tenth aspect of the invention.

In a twelfth aspect the invention provides a cell incorporating a cellmarker of the tenth aspect of the invention.

In a thirteenth aspect the invention provides a cell produced by themethod of the eleventh aspect of the invention.

In the context of the description and claims: “BODIPY” means thecompound assigned the Chemical Abstracts Service (CAS) Registry number138026-71-8 and the CA index name: Boron,difluoro[2-[(2H-pyrrol-2-ylidene-κN)methyl]-1H-pyrrolato-κN]-,(T-4)-(9CI); “fluorescein” means the chemical structure assigned theChemical Abstracts Service (CAS) Registry number 518-47-8 and the CAindex name: Spiro[isobenzofuran-1(3H),9′-[9H] xanthen]-3-one,3′,6′-dihydroxy-, sodium salt (1:2); “fluorophore” means thesubstructure or portion of a fluorescent molecule to which thefluorescent properties of the molecule are attributed, and “or one ofits derivatives” means a chemical modification of the chemical structureto provide a fluorophore with substantially equivalent physico-chemicalproperties, but modified spectral characteristics.

DETAILED DESCRIPTION

The synthetic molecule constructs of the invention spontaneously andstably incorporate into a lipid bi-layer, such as a membrane, when asolution of the molecule is contacted with the lipid bi-layer. Whilstnot wishing to be bound by theory it is believed that the insertion intothe membrane of the lipid tails of the lipid (L) is thermodynamicallyfavoured. Subsequent disassociation of the synthetic molecule constructfrom the lipid membrane is believed to be thermodynamically unfavoured.Surprisingly, the synthetic molecule constructs identified herein havealso been found to be water soluble.

The synthetic molecule constructs of the invention are used to transformcells resulting in qualitative and/or quantitative changes in thesurface antigens expressed. It will be recognised that thetransformation of cells in accordance with the invention isdistinguished from transformation of cells by genetic engineering. Theinvention provides for phenotypic transformation of cells withoutgenetic transformation.

In the context of this description the term “transformation” inreference to cells is used to refer to the insertion or incorporationinto the cell membrane of exogenously prepared synthetic moleculeconstructs thereby effecting qualitative and quantitative changes in thecell surface antigens expressed by the cell.

The synthetic molecule constructs of the invention comprise an antigen(including fluorophores) (F) linked to a lipid portion (or moiety) (L)via a spacer (S₁—S₂). The synthetic molecule constructs can be preparedby the condensation of a primary aminoalkyl, secondary aliphaticaminoalkyl or primary aromatic amine derivative of the antigen with anactivated lipid.

Methods of preparing neoglycoconjugates have been reviewed (Bovin(2002)).

A desired phenotypic transformation may be achieved using the syntheticmolecule constructs of the invention in a one-step method or a two-stepmethod. In the one step method the water soluble synthetic moleculeconstruct (F—S₁—S₂-L) comprises the surface antigen as F.

In the two-step method the synthetic molecule construct (F-S₁-S₂-L)comprises an antigen (F) that serves as a functional group to which asurface antigen can be linked following insertion of the syntheticmolecule construct into the membrane. The functional group can be agroup such as a lectin, avidin or biotin. When used in the two stepmethod the synthetic molecule construct is acting as a syntheticmembrane anchor.

In accordance with the invention the primary aminoalkyl, secondaryaliphatic aminoalkyl or primary aromatic amine and the activator of thelipid are selected to provide a synthetic molecule construct that iswater soluble and will spontaneously and stably incorporate into a lipidbi-layer when a solution of the synthetic molecule construct iscontacted with the lipid bi-layer.

In the context of this description the phrase “water soluble” means astable, single phase system is formed when the synthetic moleculeconstruct is contacted with water or saline (such as PBS) in the absenceof organic solvents or detergents, and the term “solution” has acorresponding meaning.

In the context of this description the phrase “stably incorporate” meansthat the synthetic molecule constructs incorporate into the lipidbi-layer or membrane with minimal subsequent exchange between the lipidbi-layer or membrane and the external aqueous environment of the lipidbi-layer or membrane.

The selection of the primary aminoalkyl, secondary aliphatic aminoalkylor primary aromatic amine and the activator depends on thephysico-chemical properties of the antigen (F) to be linked to the lipid(L).

It will be understood by those skilled in the art that for anon-specific interaction, such as the interaction between a diacyl- ordialkyl-glycerolipid and a membrane, structural and stereo-isomers ofnaturally occurring lipids can be functionally equivalent. For example,it is contemplated by the inventors that diacylglycerol 2-phosphatecould be substituted for phosphatidate (diacylglycerol 3-phosphate).Furthermore, it is contemplated by the inventors that the absoluteconfiguration of phosphatidate can be either R or S.

The inventors have determined that to prepare synthetic moleculeconstructs (F-S₁-S₂-L) of the invention where the antigen (F) is acarbohydrate (or other antigen) with similar physico-chemical propertiesto the oligosaccharide of the A-, B- or H-antigens of the ABO bloodgroups and the lipid moiety (L) is a glycerophospholipid, S₁ is selectedfrom —O(CH₂)_(n)NH— and S₂ is selected from —CO(CH₂)_(n)CO— or—CO(CH₂)_(m)NHCO(CH₂)_(n)CO— (where n and m are independently 2 to 5).

It will be understood by one skilled in the art that once the structureof the spacer (S₁—S₂) has been determined for a given class of antigens,e.g. carbohydrates, the same structure of the spacer can be adopted toprepare synthetic molecule constructs of other classes of antigen, e.g.fluorophores, with similar physico-chemical properties.

The structure of the spacer for synthetic molecule constructs(F-S₁-S₂-L) of the invention where F is a glycotope of the A-, B- andH-antigens of the ABO blood groups, may be the structure of the spacerselected to prepare synthetic molecule constructs comprising otherglycotopes with physico-chemical properties similar to the glycotopes ofthe A-, B- and H-antigens of the ABO blood groups.

In principle the glycotope of a broad range of blood group relatedglycolipids or glycoproteins could be the antigen (F) of the syntheticmolecule construct F-S₁-S₂-L where S₁—S₂-L is identical or equivalent tothe corresponding portion of the synthetic molecule constructsdesignated A_(tri)-sp-Ad-DOPE (I), A_(tri)-spsp₁-Ad-DOPE (II),A_(tri)-sp-Ad-DSPE (III), B_(tri)-sp-Ad-DOPE (VI), H_(tri)-sp-Ad-DOPE(VII), H_(di)-sp-Ad-DOPE (VIII), Galβ-sp-Ad-DOPE (IX),Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc-sp-Ad-DOPE (XII), andFucα1-2Galβ1-3(Fucα1-4)GlcNAc-sp-Ad-DOPE (XIII).

TABLE 1 The structures (where G1 is D-Gal, Gc is D-Glc, GcN is D-GlcNAc,M is D-Man, F is L-Fuc and NA is NeuAc) of known blood group-relatedglycolipids and glycoproteins. Glycolipids A-12-2

A-14-2

A-16-2

A-6-1

A-6-2

A-7-1 (ALe^(b))

A-7-2 (ALe^(y))

A-7-4

A-8-2

A-9-3

B-12-2

B-14-2

B-16-2

B-6-1

B-6-2

B-8-2

BLe^(b)-7

BLe^(y)-7

Cad erythrocyte

Cad hepato- carcinoma antigen

Disialoyl Le^(a)-7

Erythrocyte FI antigen

Forssman antigen GalNAcα1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1→1CerGloboside/P GalNAcβ1→3Galα1→4Galβ1→4Glcβ1→1Cer GlobotriaosylceGalα1→4Galβ1→4Glcβ1→Cer ramide/P^(K) H-5-1

H-5-2

H-6-4

H-8-3

Hematoside/G_(M3) NeuAcα2→3Galβ1→4Glcβ1→1Cer I antigen

i antigen/lacto- Galβ1→4GlcNAclβ1→3Galβ1→4GlcNAcβ1→3Galβ1→4Glcβ1→1CerN-nor- hexaosylceramide I-active polyglycosylcera- mide

Lactosylceramide Galβl→4Glcβ1→Cer LactotriaosylGlcNAcβ1→3Galβ1→4Glcβ1→Cer ceramide Le^(a)-11

Le^(a)-5

Le^(b)-6

Le^(b)-8

Le^(c)- Galβ1→3GlcNAcβ1→3Galβ1→4Glcβ1→1Cer 4/Lactotetraosyl ceramideLe^(c)-9 (fucosylated backbone)

Le^(c)-9 (fucosylated branch)

Le^(x)-11

Le^(x)-5

Le^(x)-7

Le^(x)-8

Le^(y)-6

LKE/′GL 7/SSEA-4 NeuAcα2→3Galβ1→3GalNAcβ→3Galα1→4Galβ1→4Glcβ1→1Cer P₁Galα1→4Galβ1→4GlcNAcβ1→3Galβ1→4Glcβ1→1Cer Paragloboside/Galβ1→4GlcNAcβ1→3Galβ1→4Glcβ1→1Cer neolactotetrao- sylceramide P-likeGalNAcβ1→3Ga1β1→4GlcNAcβ1→3Galβ1→4Glcβ1→1Cer SialoylNeuAcα2→3Galβ1→4GlcNAcβ1→3Galβ1→4Glcβ1→1Cer paragloboside/sialoylneolacto- tetraosylceramide Sialyl Le^(a)- 6/gastrointestinal\cancer antigen (GICA or Ca 19-9)

Sialyl Le^(x)

Sialyl-nor-hexaosyl-NeuAcα2→3Galβ1→4GlcNAcLβ1→3Galβ1→4GlcNAcβ1→3Galβ1→4Glcβ1→Cer ceramide/sialoyl-lacto-N-nor- hexaosylceramide VIM-2

O-linked Glycoproteins Cad oligosaccharide

Disialotetrasac charide

Disialoyl group oligosaccharide

GlcNAc oligosaccharide

H-active trisaccharide

Monosialotrisac charide

Mucin oligosaccharide/ A-active glycoprotein

Ovarian cyst A- active glycoprotein-18

Ovarian cyst A- active glycoprotein-6a

Ovarian cyst A- active glycoprotein-6b

Ovarian cyst Le^(a)-active glycoprotein-10

Ovarian cyst Le^(a)-active glycoprotein-7

Sialylated H-active tetrasaccharide

N-linked Glycoproteins Adult lactosaminoglycan

Complex type/Alkali-stable chain

Difucosyl adult erythrocyte antigen

Difucosyl foetal erythrocyte antigen

Disialyl foetal erythrocyte antigen

Foetal lactosaminoglycan

High-mannose type

Hybrid type

Monofucosyl- disialyl foetal erythrocyte antigen (disialyl group onbranch)

Monofucosyl- monosialoyl adult erythrocyte antigen

Monofucosyl- monosialoyl adult erythrocyte antigen

Monofucosyl- monosialyl foetal erythrocyte antigen (fucosylatedbackbone)

Monofucosyl- monosialyl foetal erythrocyte antigen (fucosylated branch)

Tamm-Horsfall glycoprotein

Trisialoyl foetal erythrocyte antigen (disialoyl group on branch)

In general, for almost all examples of A-antigens the terminal A sugarN-acetylgalactoseamine (GalNAc) can be replaced with the B sugargalactose (Gal). Additionally, the lack of either the A or B determinantcreates the equivalent H determinant.

It will be understood by those skilled in the art that the syntheticmolecule constructs (F-S₁-S₂-L) of the invention where F is anoligosaccharide may be used as “synthetic glycolipids” and substitutedfor glycolipids obtained from biological (botanical or zoological)sources.

In the context of this description of the invention the term“glycolipid” means a lipid containing carbohydrate of amphipathiccharacter including: glycosylated glycerolipids, such as glycosylatedphosphoglycerides and glycosylglycerides; glycosylated sphingolipids(neutral glycolipids) such as glycosylceramides or cerebrosides; andgangliosides (acidic glycolipids).

In the context of this description of the invention the phrase“glycolipid-linked antigen” means a lipid containing carbohydrate inwhich an antigen (e.g. a protein) is linked to the glycolipid via thecarbohydrate portion of the molecule. Examples of glycolipid-linkedantigens include GPI-linked proteins.

It will be understood by those skilled in the art that a glycolipid isitself an antigen. The term and phrase “glycolipid” and“glycolipid-linked antigen” are used to distinguish between naturallyoccurring molecules where the antigen is the glycolipid and naturallyoccurring molecules where the antigen is linked to the glycolipid viathe carbohydrate portion of the glycolipid. By analogy the syntheticmolecule constructs of the invention could be described as both“synthetic glycolipids” and “synthetic membrane anchors” to the extentthat the antigen may be the synthetic glycolipid per se or attached tothe synthetic glycolipid.

It will be understood by those skilled in the art that the carbohydrateportion of a glycolipid may be modified and linked to other antigens bythe methods described in the specification accompanying theinternational application no. PCT/NZ2003/00059 (publ. no. WO 03/087346).

In the context of this description of the invention the term “glycotope”is used to refer to the antigenic determinant located on thecarbohydrate portion of a glycolipid. The classification of glycolipidantigens in blood group serology is based on the structure of thecarbohydrate portion of the glycolipid.

In blood group serology it is known that the terminal sugars of theglycotopes of A-antigens are GalNAcα1-3(Fucα1-2)Galβ, and the terminalsugars of the glycotopes of the B-antigens are Galα1-3(Fucα1-2)Gals.Incorporation into the membrane of RBCs of water soluble syntheticmolecule constructs of the invention where F is GalNAcα1-3(Fucα1-2)Galβor Galα1-3(Fucα1-2)Galβ provides RBCs that are serologically equivalentto A-antigen or B-antigen expressing RBCs, respectively.

The terminal three sugars of the carbohydrate portion of the naturallyoccurring A- or B-antigen are the determinant of the A and B bloodgroupings. The terminal four or five sugars of the carbohydrate portionof the naturally occurring A-antigen are the determinant of the A bloodsub-groupings A type 1, A type 2, etc. Accordingly, the RBCsincorporating the synthetic molecule constructs of the invention can beused to characterise and discriminate between blood typing reagents(antibodies) of differing specificity.

Water soluble synthetic molecule constructs of the invention thatexclude a carbohydrate portion are contemplated by the inventors.Antigens other than carbohydrates or oligosaccharides, but with similarphysico-chemical properties, may be substituted for F in the “syntheticglycolipids” described.

Synthetic molecule constructs of the invention that comprise an antigen(F) with differing physico-chemical properties to those of carbohydratesor oligosaccharides are also contemplated by the inventors. Watersoluble synthetic molecule constructs comprising these antigens may beprepared by selecting different spacers.

The advantages provided by the synthetic molecule constructs of thisinvention will accrue when used in the practice of the inventionsdescribed in the specifications accompanying international applicationnos. PCT/NZ02/00212 (publ. no. WO 03/034074) and PCT/NZ03/00059 (publ.no. WO 03/087346). The disclosures of the specifications accompanyingthese applications are incorporated herein by reference.

The synthetic molecule constructs overcome many of the limitations ofusing natural glycolipids in the practice of these inventions. Aparticular advantage of the synthetic molecule constructs is theirsuperior performance and ability to be used in the transformation ofcells at reduced temperatures, e.g. 4° C.

As described herein not all structures of the spacer (S₁—S₂) willprovide a synthetic molecule construct (F-S₁-S₂-L) that is water solubleand spontaneously and stably incorporate into a lipid bi-layer such as acell membrane. The synthetic molecule constructs designatedA_(tri)-sp-lipid (IV) and A_(tri)-PAA-DOPE (V) were determined not to bewater soluble and/or unable to spontaneously and stably incorporate intoa lipid bilayer such as a cell membrane.

where x and y are in the range 0.05 to 0.2

The invention will now be illustrated by reference to the followingnon-limiting examples and figures of the accompanying drawings in which:

FIG. 1 shows Diamed results of Cellstab™ stored cells transformed bynatural A glycolipid transformation solution at (L to R) 10 mg/mL, 5mg/mL, 2 mg/mL, 2 mg/mL* and 1 mg/mL. Antisera used are Albaclone (top)and Bioclone (bottom). (*-transformation solution (containingglycolipids) was not washed out after the incubation, it was left inover night and washed out the next day (day 2).)

FIG. 2 shows Diamed results of Cellstab™ stored cells transformed bynatural B glycolipid transformation solution at (L to R) 10 mg/mL, 5mg/mL, 2 mg/mL, 2 mg/mL* and 1 mg/mL. Antisera used are Albaclone (top)and Bioclone (bottom). (*-transformation solution (containingglycolipids) was not washed out after the incubation, it was left inover night and washed out the next day (day 2)).

FIG. 3 shows FACS analysis following in vitro transformation of humanLe(a-b-) red cells with natural Le^(b)-6 glycolipid over time at threetransformation temperatures, 37° C. (top), 22° C. (middle) and 4° C.(bottom).

FIG. 4 shows Diamed results of cells transformed at 4° C. byA_(tri)-sp-Ad-DOPE (I) transformation solution at (L to R): washed 0.08mg/mL; unwashed 0.08 mg/mL; washed 0.05 mg/mL; unwashed 0.05 mg/mL;washed 0.03 mg/mL; and unwashed 0.03 mg/mL. The antisera used wasBioclone anti-A.

FIG. 5 shows cells that were no longer washed prior to testing. Diamedresults of cells transformed at 4° C. by A_(tri)-sp-Ad-DOPE (I)transformation solution at (L to R): 0.08 mg/mL, 0.05 mg/mL and 0.03mg/mL. The antisera used was Bioclone anti-A.

FIG. 6 shows in the left column Diamed results of cells transformed at4° C. by B_(tri)-sp-Ad-DOPE (VI) transformation solution at (L to R):washed 0.6 mg/mL; unwashed 0.6 mg/mL; washed 0.3 mg/mL; unwashed 0.3mg/mL; washed 0.15 mg/mL; and unwashed 0.15 mg/mL; and in the rightcolumn Diamed results of cells transformed at 4° C. byB_(tri)-sp-Ad-DOPE (VI) transformation solution at (L to R): washed 0.08mg/mL; unwashed 0.08 mg/mL; washed 0.05 mg/mL; unwashed 0.05 mg/mL;washed 0.03 mg/mL; and unwashed 0.03 mg/mL. The antisera used wasBioclone anti-B.

FIG. 7 shows cells that were no longer washed prior to testing. Diamedresults of cells transformed at 4° C. by B_(tri)-sp-Ad-DOPE (VI)transformation solution at (L to R): 0.6 mg/mL, 0.3 mg/mL and 0.15mg/mL.

FIG. 8 shows Diamed results of cells transformed at 4° C. by paralleltransformation with A_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI).Wells 1 and 2 (L to R) contain washed A 0.07+B 0.3 mg/mL against anti-Aand anti-B. Wells 3 and 4 contain unwashed A 0.07+B 0.3 mg/mL againstanti-A and anti-B.

FIG. 9 shows cells that were no longer washed prior to testing. Diamedresults of cells transformed at 4° C. by parallel transformation withA_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI). Wells 1 and 2 (L toR) contain unwashed A 0.07+B 0.3 mg/mL against anti-A and anti-B.

FIG. 10 shows Diamed results of cells transformed at 4° C. by paralleltransformation with A_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI).Wells 1 and 2 (L to R) contain washed A 0.07+B 0.2 mg/mL against anti-Aand anti-B. Wells 3 and 4 contain unwashed A 0.07+B 0.2 mg/mL againstanti-A and anti-B.

FIG. 11 shows cells that were no longer washed prior to testing. Diamedresults of cells transformed at 4° C. by parallel transformation withA_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI). Wells 1 and 2 (L toR) contain unwashed A 0.07+B 0.2 mg/mL against anti-A and anti-B.

FIG. 12 shows Diamed results of cells transformed at 4° C. by paralleltransformation with A_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI).Wells 1 and 2 (L to R) contain washed A 0.06+B 0.3 mg/mL against anti-Aand anti-B. Wells 3 and 4 contain unwashed A 0.06+B 0.3 mg/mL againstanti-A and anti-B.

FIG. 13 shows cells that were no longer washed prior to testing. Diamedresults of cells transformed at 4° C. by parallel transformation withA_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI). Wells 1 and 2 (L toR) contain unwashed A 0.06+B 0.3 mg/mL against anti-A and anti-B.

FIG. 14 shows Diamed results of cells transformed at 4° C. by paralleltransformation with A_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI).Wells 1 and 2 (L to R) contain washed A 0.06+B 0.2 mg/mL against anti-Aand anti-B. Wells 3 and 4 contain unwashed A 0.06+B 0.2 mg/mL againstanti-A and anti-B.

FIG. 15 shows cells that were no longer washed prior to testing. Diamedresults of cells transformed at 4° C. by parallel transformation withA_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI). Wells 1 and 2 (L toR) contain unwashed A 0.06+B 0.2 mg/mL against anti-A and anti-B.

FIG. 16 shows Diamed results of cells transformed at 4° C. by paralleltransformation with A_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI).Wells 1 and 2 (L to R) contain washed A 0.05+B 0.3 mg/mL against anti-Aand anti-B. Wells 3 and 4 contain unwashed A 0.05+B 0.3 mg/mL againstanti-A and anti-B.

FIG. 17 shows cells that were no longer washed prior to testing. Diamedresults of cells transformed at 4° C. by parallel transformation withA_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI). Wells 1 and 2 (L toR) contain unwashed A 0.05+B 0.3 mg/mL against anti-A and anti-B.

FIG. 18 shows Diamed results of cells transformed at 4° C. by paralleltransformation with A_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI).Wells 1 and 2 (L to R) contain washed A 0.05+B 0.2 mg/mL against anti-Aand anti-B. Wells 3 and 4 contain unwashed A 0.05+B 0.2 mg/mL againstanti-A and anti-B.

FIG. 19 shows cells that were no longer washed prior to testing. Diamedresults of cells transformed at 4° C. by parallel transformation withA_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI). Wells 1 and 2 (L toR) contain unwashed A 0.05+B 0.2 mg/mL against anti-A and anti-B.

FIG. 20. Red blood cells following contact with a synthetic moleculeconstruct (XVI) viewed with a fluorescence microscope at 470 nm under250× magnification.

COMPARATIVE EXAMPLES

The Comparative Examples do not form part of the invention claimed. TheComparative Examples describe red blood cell (RBC) transformation withnatural glycolipids.

Comparative Example 1—Preparation of Natural Glycolipids

Purification by HPLC

In the first stage, columns were packed with dry silica (15-25 μm)before each run. Relatively dirty samples could be used in HPLC becausethe silica could be discarded along with the theoretically high level ofirreversibly bound contaminants.

Glycolipids were separated on silica gel with a mobile phase ofincreasing polarity. The program was a linear gradient beginning with100% chloroform-methanol-water 80:20:1 (v/v) and ending with 100%chloroform-methanol-water 40:40:12 (v/v).

The HPLC equipment used was a Shimadzu system capable of pumping andmixing four separate solvents at programmed ratios. As chloroform,methanol and water evaporate at different rates, a program was developedwhereby the solvent components were not mixed prior to entering theHPLC.

The Shimadzu HPLC mixes four different liquids by taking a “shot” fromeach of four bottles in turn. “Shots” of chloroform and water directlynext to each other in the lines may cause miscibility problems. Methanolwas sandwiched in between these two immiscible components. Additionally,the water was pre-mixed with methanol in a 1:1 ratio to further preventproblems with miscibility.

Comparative Example 2—Transformation of Red Blood Cell Transformationwith Natural Glycolipids

Agglutination

Transformation of red blood cells was assessed by agglutination usingthe Diamed-ID Micro Typing System in addition to using conventional tubeserology. Diamed ABO typing cards were not used. The cards used wereNaCl, Enzyme test and cold agglutinin cards, which were not pre-loadedwith any antisera or other reagents. This allowed the use of specificantisera with both methodologies.

A comparative trial was carried out between tube serology and the Diamedsystem to establish the performance of the two systems. Cells weretransformed at 25° C. for 4 hours. Seraclone and Alba-clone anti-A serawere used to gauge equivalency. The results are shown in Table 3 below.

TABLE 2 Antisera used in comparison of tube serology with the Diamedsystem. Manufacturer Catalogue ref Lot Expiry Albaclone, SNBTS Anti-A.Z0010770 12.12.04 Seraclone, Biotest 801320100 1310401 12.04.03

TABLE 3 Agglutination results comparing tube serology with the Diamedsystem. A glycolipid (mg/mL) 10 5 2 1 0 Tube Albaclone 3+ 2+ 0 0 0Seraclone 3+ 2+ 0 0 0 Diamed Albaclone 2+ 2+ 0 0 0 Seraclone 3+ 2+ 1+ w+0

In this experiment, the Diamed system proved to be more sensitive to theweaker reactions than tube serology with the Seraclone anti-A, but notwith Albaclone. These reagents are formulated differently and are thusnot expected to perform identically. However, the fact that theSeraclone anti-A tube serology combination did not detect positivity isprobably due to operator interpretation. The weaker reactions arenotoriously difficult to accurately score, and the difference between 1+and 0 can be difficult to discern in tubes.

Optimisation

The variables of glycolipid concentration, incubation temperature,incubation duration, diluent and storage solution were examined fortheir effect on cell health. Efficiency and stability of transformationwas assessed by agglutination with the relevant antibody.

TABLE 4 Tube serology agglutination of natural glycolipid A transformedcells over different times and temperatures. A 10 5 2 1 0.1 0.01 0.0010.0001 0 Seraclone 3+ 2+ 0  0  0 (37° C. for 1.5 hours) Seraclone 4+ 3+2+ 1+ w+ 0 0 0 0 (25° C. for 4 hours)Glycolipid Concentration

Initial transformation experiments were carried out with a highlypurified (HPLC) Le^(b) glycolipid sample and a less pure blood group Aglycolipid sample.

Transformation was performed at 37° C. for 1.5 hours The A glycolipidsample contained other lipid impurities and thus comparatively lessblood group A molecules by weight than the Le^(b) glycolipid sample ofequivalent concentration (w/v). This seems to be borne out by the factthat higher concentrations of the A glycolipid than the Le^(b)glycolipid were required to produce equivalent agglutination scores (seeTable 6).

The level of impurity in the A glycolipid sample may also havecontributed to the lower stability over the 62 day period—theA-transformed cells ‘died’ at the highest concentration (having receivedthe largest dose of impurity).

TABLE 5 Anti-A and anti-Le^(b) used in initial testing of naturalglycolipid transformation. Manufacturer Catalogue ref Batch numberExpiry Anti-A 801320100 1310401 12.04.03 Seraclone, Biotest Anti-Le^(b)12801 CSL

TABLE 6 Stability of RBCs transformed with natural A and Le^(b)glycolipid as assessed by tube serology agglutination over the period of62 days. Glycolipid Le^(b) A (mg/mL) Day 1 Day 25 Day 62 Day 1 Day 25Day 62 10 4+ 2-3+  3+ 2+ ? 5 4+ 2-3+  2+ 2+ w+ 2 3+ 1-2+ 0 1+ 0 1 4+  2+0 1+ 0 0.1 3+ 2+ 0 0 0.01 2+ 2+ 0 0 0.001 2+ 2+ 0 0 0.0001 2+ 0  0 0 00  0  0 0 0  0

The above cells were also rated for haemolysis and these results areshown in Table 7 below.

TABLE 7 Haemolysis as assessed visually. Glycolipid Haemolysisconcentration Le^(b) A (mg/mL) Day 1 Day 25 Day 62 Day 1 Day 25 Day 6210 h 0 h h h dead 5 hh 0 hhh w 0 hh 2 w 0 hhh w 0 hhhhh 1 w 0 hhh h 0hhhh 0.1 h hhh 0.01 hh 0.001 h 0.0001 h Control h 0 h h h Day 1—in thesupernatant of the first wash after transformation; Days 25 and 62—inthe cell preservative solution before the cells are resuspended afterstorage. Scoring scale is analogous to the 4+ to 0 agglutination scale:hhhh—severely haemolysed, hhh—very haemolysed, hh—moderately haemolysed,h—mildly haemolysed, w—faintly haemolysed and 0—no haemolysis seen.

These results show that cell haemolysis can be shown to be associatedwith transformation with high concentrations of glycolipid. It isunclear whether the mechanism underlying this is disruption of theplasma membrane by large amounts of glycolipid being inserted, the rateof that insertion, or is possibly due to the quantity of associatedimpurity. However, the results for Le^(b) at day 62 seem to support thefirst explanation.

The Le^(b) sample was highly purified—before being dissolved, it was apowder of pure white colour, and thus it is unlikely that the haemolysiswas due to the deleterious effect of impurities. It is clear to see thatat 62 days, the amount of haemolysis occurring diminishes in line withthe decrease in the glycolipid concentration.

Incubation Temperature

Experiments were carried out to investigate other possible mechanismsfor the reduction of haemolysis of RBCs during the insertion step.Previous experiments had shown that haemolysis was worse at higherglycolipid concentrations than at lower concentrations, and it isthought that haemolysis may also be related to the rate of glycolipidinsertion. Since temperature is believed to affect the rate ofinsertion, experiments were conducted comparing transformation at 37° C.with transformation at room temperature (RT; 25° C.).

Since the rate was expected to slow down as temperature decreased, theincubation period for the RT experiment was 4 hrs. Haemolysis wasassessed visually and scored following insertion. Serology tests werealso performed on the cells. The results are shown in Table 8.

TABLE 8 The effect of incubation temperature on haemolysis andagglutination during insertion of glycolipids into RBC membranes.Haemolysis was scored visually at each of the three washes Haemolysis RT37° C. Glycolipid wash wash wash wash wash wash Serology (mg/mL) 1 2 3 12 3 RT 37° C. 10 w 0 0 hh w 0 2+ 2+ 1 w 0 0 hh h vw 1+ w+Incubation Duration

Incubation at 37° C. was carried out for 1 and 2 hours and its effect oncell health and transformation assessed by agglutination with therelevant antibody.

TABLE 9 Antisera used in the duration of incubation trial. ManufacturerCatalogue ref Batch number Expiry date Albaclone, SNBTS Anti-A. Z001077012.12.04 Bioclone, OCD Anti-A, DEV01102 — experimental reagentAlbaclone, SNBTS Anti-B Z0110670 01.07.05 Bioclone, OCD Anti-B, DEV01103— experimental reagent

TABLE 10 Effect of incubation time on agglutination of cells transformedwith natural glycolipids. Concentration Albaclone BioClone Glycolipid(mg/mL) 1 hour 2 hours 1 hour 2 hours A 10 4+ 4+ 4+ 4+ 5 4+ 4+ 4+ 2+ 24+ 3+ 3+ 2+ 1 3+ 2+ 2+ 2+ 0.5 2+ 2+ 1+ w+ B 10 3+ 2+ 4+ 1+ 5 3+ 2+ 3+ 2+2 2+ 2+ 2+ 1+ 1 1+ w+ 1+ w+ 0.5 1+ w+ w+ w+

These results indicate that increasing the duration of incubation duringnatural glycolipid insertion does not enhance agglutination. In fact,the agglutination scores are reduced after the two hour incubation. Thismay be due to the destabilisation of the membrane or exchange of theglycolipids back into solution.

Diluent

Experiments were also carried out to determine if changing theglycolipid diluent solution could reduce haemolysis. Working strengthPBS was compared with 2×PBS and 2% Bovine Serum Albumin (BSA) in workingstrength PBS. Cells were incubated at 37° C. for 1.5 hours. The resultsare shown in Table 11.

TABLE 11 Study on the effect on haemolysis of changing the glycolipiddiluent solutions during insertion of glycolipids into RBC membranes.Glycolipid concentration Glycolipid Diluent Solution (mg/mL) PBS 2 × PBS2% BSA in PBS 40 Hhh hhh hhh 30 Hhh hhh hhh 20 Hhh hhh hhh 10 Hhh hhhhhh 0 0 0 0Stability

Once A and B blood group glycolipids had been HPLC purified to anacceptable level, an experiment to find the appropriate concentrationsfor stability trials was carried out.

TABLE 12 Early stability trial of cells transformed with natural Aglycolipid. A Expt Day 10 5 2 1 0.1 0.01 0.001 0.0001 0 1 7 4+ 3-4+  1+0 0 0 0 0 0 2 43 3+ w+ 0 0 0 0 0 0 0 3 50 1+ 0  0 0 4 60 3+ 1+ 0 5 67 w+vw vw 6 74 2+ 0  0 7 81 2+ 1+ 0

TABLE 13 Antisera used in stability trials (Table 14 and Table 15).Batch Expiry Manufacturer Catalogue ref number date Albaclone, SNBTSAnti-A. Z0010770 12.12.04 Bioclone, OCD Anti-A, experimental reagentDEV01102 — Albaclone, SNBTS Anti-B Z0110670 01.07.05 Bioclone, OCDAnti-B, experimental reagent DEV01103 —

TABLE 14 Tube serology of O RBCs transformed with A glycolipid in orderto establish appropriate concentrations for stability trials.Transformation at 25° C. for 4 hours. A glycolipid (mg/mL) Anti-A Expt10 5 2 1 0.5 0.1 0.01 0.001 0 Alba 1 3+ 2+ 1+ 0  0 0 0 0 2 4+ 4+ 3+ 2+w+ Bio 1 3+ 2+ 1+ 0  0 0 0 0 2 4+ 4+ 3+ 2+ w+

TABLE 15 Tube serology of O RBCS transformed with B glycolipid in orderto establish appropriate concentrations for stability trials. Bglycolipid (mg/mL) Anti-B Expt 10 5 2 1 0.5 0.1 0.01 0.001 0 Alba 1 2+1+ w+ 0 0 0 0 0 2 1+ 1+ w+ 0 w+ Bio 1 3+ 2+ w+ 0 0 0 0 0 2 1+ 1+ w+ 0 w+

Two sets of cells were transformed with different concentrations ofnatural A glycolipid. Transformation was performed at 25° C. One set ofcells was tested long term, and one set of cells was tested weekly foragglutination. The agglutination results from tube serology and Diamedare shown in Table 16 below. All cells were stored in Cellstab™ inbottles with flat bases. The cells showed minimal to no haemolysis atany time.

TABLE 16 Agglutination results for cells transformed with differentconcentrations of natural A glycolipid. Results were obtained usingAlbaclone anti-A * - Albaclone, while all others used Seraclone anti-A.A glycolipid (mg/mL) 10 5 2 1 0.1 control Long term testing Day 1 Tube4+ 3+ 2+ 1+ +w 0 Diamed 3+ 3+ +w 0 0 0 Day 17 Tube 3+ 2+ 0 0 0 Diamed 3+2+ 1+ 0 0 Weekly testing Day 1 Tube 3+ 2+ 0 0 Diamed 3+ 0 0 0 Day 8 Tube1+ 0 0 0 Diamed 3+ 0 0 0 Day 15 Tube 1+ 0 0 0 Diamed 3+ 2+ 0 0 Day 22Tube 3+ 0 0 0 Diamed 3+ 0 0 0 Day 29 Tube *+w *0 *0 *0 Diamed *3− *0 *0*0 Day 36 Tube * * * *0 Diamed *3− *0 *0 *0 Day 43 Tube * * * *0Diamed * * * *0Storage Solution

Comparison of the two cell storage solutions, Celpresol™ (CSL) andCellstab™ (Diamed) was carried out to test their relative abilities tosupport modified RBCs.

The stability of RBCs transformed with blood group A and B antigensolutions of varying concentrations when stored in two different cellpreservative solutions—Cellstab™ and Alsevers™—was trialed.

A and B antisera from two different sources were used in serologytesting.

All cells were tested using the standard tube serology platform up to 42days, at which time the cell agglutination reactions had become toodifficult to score manually (see Table 17 for A results and Table 18 forB results).

Diamed gel-card testing was carried out to day 56 for the Alseversstored cells, and discontinued at day 63 due to fungal contamination(although still returning positive scores). The Cellstab™ stored cellscontinued to be tested up to day 70, and were still viable at this point(see FIG. 1 for A results and FIG. 2 for B results).

The reagents used in the stability trial are shown in Table 13.

TABLE 17 Tube serology results of stability trial of cells transformedwith varying concentrations of A glycolipid and stored in eitherCellstab ™ or Alsevers ™. Albaclone Anti-A Bioclone Anti-A Cell (SNBTS)Transformation (OCD - Developmental storage Solution (mg/mL) reagent)Day solution 10 5 2 2* 1 10 5 2 2* 1 2 Alsevers 4+ 3+ 2+ 1+ w+ 3+ 3+ 1+1+ 0 Cellstab ™ 4+ 4+ 3+ 1+ 1+ 3+ 3+ 2+ 1+ 0 8 Alsevers 4+ 4+ 2+ 1+ 1+2+ 2+ 1+ 1+ 0 Cellstab ™ 4+ 4+ 3+ 2+ 1+ 3+ 3+ 2+ w+ 0 14 Alsevers 4+ 3+2+ 2+ w+ 2+ 1+ w+ vw 0 Cellstab ™ 4+ 3+ 3+ 2+ w+ 3+ 2+ w+ vw 0 21Alsevers 3+ 2+ 2+ 2+ 1+ 2+ 2+ 2+ 1+ 0 Cellstab ™ 3+ 3+ 2+ + ^(‡) 2+ ^(‡)^(‡) ^(‡) 0 28 Alsevers 2+ 2+ 1+ 1+ 0  2+ 2+ 1+ 1+ 0 Cellstab ™  2+^(‡) 2+^(‡) ^(‡) ^(‡) 0  1+ w+ 0  0  0 36 Alsevers 3+ 2+ 2+ 2+ 1+ 3+ 3+ 2+1+  1+ Cellstab ™  3+^(‡)  2+^(‡) ^(‡) ^(‡) ^(‡)  3+^(‡) ^(‡) ^(‡) ^(‡)^(‡) 42 Alsevers 3+ 3+ 1+ w+ 0  2+ 2+ 2+ 1+  1+ Cellstab ™  4+^(‡) 4+^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) 0 *transformation solution(containing glycolipids) was not washed out after the incubation, it wasleft in over night and washed out the next day. ^(‡)positive cellbutton, but cells fall off as negative (score assignment impossible).FACS Analysis of Glycolipid Insertion

Transformation of human Le(a-b-) red cells with natural Le^(b)-6glycolipid over time at three transformation temperatures (37° C., 22°C. and 4° C.) was performed (FIG. 3). Natural Le^(b)-6 glycolipid wasdissolved in plasma and used to transform RBCs at a final concentrationof 2 mg/mL and a final suspension of 10%.

TABLE 18 Tube serology results of stability trial of cells transformedwith varying concentrations of B glycolipid and stored in eitherCellstab ™ or Alsevers ™. Albaclone Anti-B Bioclone Anti-B Cell (SNBTS)Transformation (OCD - Developmental storage Solution (mg/mL) reagent)Day solution 10 5 2 2* 1 10 5 2 2* 1 2 Alsevers 3+ 3+ 1+ 1+  1+ 2+ 1+ 1+1+ 0 Cellstab ™ 3+ 3+ 2+ 2+  1+ 2+ 2+ 2+ 1+ w+ 8 Alsevers 1+ 1+ w+ 0  00  0  0  0  0 Cellstab ™ 2+ 1+ w+ 0 1+ 1+ w+ 0  0 14 Alsevers 2+ 2+ 0 w+ 0 0  1+ 1+ 2+ 0 Cellstab ™ 1+ w+ 0  0  0 2+ 2+ w+ 1+  1+ 21 Alsevers^(‡) ^(‡) ^(‡) ^(‡) ^(‡) 1  1  ^(‡) ^(‡) ^(‡) Cellstab ™ ^(‡) ^(‡) ^(‡)^(‡) ^(‡) + + + ^(‡) ^(‡) 28 Alsevers 2+ 1+ w+ 0  0 2+ 1+ 2+ 0  0Cellstab ™ ^(‡) ^(‡) ^(‡) 0  0 ^(‡) 0  ^(‡) ^(‡) 0 36 Alsevers 2+ 2+ 2+1+  1+ 2+ 2+ 2+ 1+  1+ Cellstab ™ ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡)^(‡) ^(‡) ^(‡) 42 Alsevers 2+ 2+ 2+ 2+ w+ 2+ 2+ 1+ w+ w+ Cellstab ™ ^(‡)^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) *transformation solution(containing glycolipids) was not washed out after the incubation, it wasleft in over night and washed out the next day. ^(‡) positive cellbutton, but cells fall off as negative (score assignment impossible).

Reactivity was determined by FACS analysis using a Gamma anti-Leb. (Theserological detection level is around 102 molecules. The insertion ofnatural glycolipids at 4° C. for 8 hours was not detectable byagglutination with antibodies.) Projection of the rate of insertioncurve from FACS analysis did not indicate that the rate of insertion at4° C. would have reached agglutination detection levels within 24 hours.

Low Incubation Temperature

Transformation of RBCs with natural A or B glycolipid was performed at37° C. for 1 hour and 2° C. for varying intervals. Cells wereagglutinated with Bioclone anti-A or Bioclone anti-B. The results areprovided in Tables 19 and 20.

TABLE 19 Diamed results of comparison of natural A glycolipidtransformation at 37° C. for 1 hour and 2° C. for varying intervals.Time Nat A (mg/mL) Temp (hours) 10 5 2 1 0 37° C. 1 3+ 3+ 2-3+  2+ 0  2°C. 1 0  0  0 0 0 4 0  0  0 0 0 8 1-2+ 0  0 0 0 24 2-3+ 2+ 1-2+ 0 0 48 3+2-3+ 2-3+ 0 0 72 3-4+ 3+  2+ 0 0

TABLE 20 Diamed results of comparison of natural B glycolipidtransformation at 37° C. for 1 hour and 2° C. for varying intervals.Time Nat B (mg/mL) Temp (hours) 10 5 2 1 0 37° C. 1 3+ 2-3+  2+ 0 0  2°C. 1 0  0 0 0 0 4 0  0 0 0 0 8 0  0 0 0 0 24 1+ 0 0 0 0 48 2+ 1-2+ 0 0 072 2+  1+ 0 0 0

The rate of transformation is slow for both natural A glycolipid andnatural B glycolipid as demonstrated by the negative agglutinationscores after 1 hour at 2° C. Considerable insertion at 37° C. for thistime interval has been demonstrated. Natural A glycolipid insertion at2° C. required 48 hours to reach the same level of insertion obtainableby transformation at 37° C. After this time further insertion was notobserved. Likewise, natural B glycolipid insertion at 2° C. was not asrapid as transformation at 37° C. The agglutination scores did notimprove upon continued incubation and thus seemed to have reachedmaximal insertion at this time point for these concentrations.

EXAMPLES

The Examples describe red blood cell transformation with the syntheticmolecule constructs of the invention. In the context of these examplesthe term “synthetic glycolipids” is used to refer to these constructs.

Example 1—Preparation of Synthetic Glycolipids

Materials and General Methods

Acetone, benzene, chloroform, ethylacetate, methanol, o-xylene, toluene,and 2-propanol were from Khimed (Russian Federation). Acetonitrile wasfrom Cryochrom (Russian Federation). DMSO, DMF, CF₃COOH, Et₃N,N,N′-dicyclohexylcarbodiimide and N-hydroxysuccinimide were from Merck(Germany). N-methylmorpholin (NMM), 2-maleimidopropionic acid anddisuccimidilcarbonate were from Fluka. Iminodiacetic acid dimethyl esterhydrochloride was from Reakhim (Russian Federation). Molecular sieves(MS 3 Å and 4 Å), trimethylsilyl trifluoromethanesulfonate, andtriphenylphosphine were from Aldrich (Germany). All hydrides,1,8-diazabicyclo[5,4,0]undec-7-ene (DBU), and trichloroacetonitrile werefrom Merck (Germany).

Anhydrous tetrahydrofuran (THF) and diethyl ether (Et₂O) were obtainedby distillation from lithium aluminium hydride (H₄AlLi). Dichloromethanefor glycoside synthesis was dried by distillation from phosphorouspentoxide and calcium hydride and stored over molecular sieves MS 4 Å.Solid reagents were dried for 2 h in vacuo (0.1 mm Hg) at 20 to 40° C.Deacetylation was performed according to Zemplen in anhydrous methanol.The solution of the acetylated compound was treated with 2 M sodiummethylate in methanol up to pH 9. When the reaction was completed, Na⁺ions were removed with cation exchange resin Dowex 50X-400 (H+) (Acros,Belgium). The solution was concentrated in vacuo.

Column chromatography was carried out on Silica gel 60 (0.040-0.063 mm,Merck, Germany). Gel chromatography was performed on Sephadex LH-20(Pharmacia, Sweden). Solvents were removed in vacuo at 30 to 40° C.Thin-layer chromatography was performed on Silica gel 60 (Merck,Germany) precoated plates. Spots were visualized by treating with 5%aqueous orthophosphoric acid and subsequent heating to 150° C. in thecase of carbohydrates or by soaking in ninhydrin solution (3 g/l in 30:1(v/v) butanol-acetic acid) in the case of amines.

Optical rotation was measured on a Jasco DIP-360 digital polarimeter at25° C. Mass spectra were recorded on a Vision-2000 (Thermo Bioanalysis,UK) MALDI-TOF mass spectrometer using dihydroxybenzoic acid as a matrix.¹H NMR spectra were recorded on a Bruker WM spectrometer (500 MHz) at25° C. Chemical shifts (δ, ppm) were recorded relative to D₂O (δ=4.750),CDCl₃ (δ=7.270), and CD₃OD (δ=3.500) as internal standards. The valuesof coupling constants (Hz) are provided. The signals in the ¹H NMRspectra were assigned by suppression of spin-spin interaction (doubleresonance) and 2D-1H, 1H-COSY experiments.

Preparation of F-S₁

Preparation of 3-aminopropyl2-acetamido-2-deoxy-α-D-galactopyranosyl-(1→3)-β-D-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside(GalNAcα1-3Galβ1-4GlcNAc-S₁) (5) (SCHEME I)

The glycosyl chloride3,4,6-tri-O-acetyl-2-azido-2-desoxy-β-D-galactopyranosylchloride (1) wasprepared according to the method disclosed in the publication of Paulsenet al (1978). The glycosyl acceptor(3-trifluoroacetamidopropyl)-2-acetamido-3-O-acetyl-6-O-benzyl-2-deoxy-4-O-(2,4-di-O-acetyl-6-O-benzyl-β-D-galactopyranosyl)-β-D-glucopyranoside(2) was prepared according to the method disclosed in the publication ofPazynina et al (2008).

A solution of the glycosyl acceptor (420 mg, 0.5 mmol), silver triflate(257 mg, 1.0 mmol), tetramethylurea (120 μl, 1.0 mmol) and freshlycalcinated molecular sieves 4 Å in dry dichloromethane (20 ml), werestirred at room temperature in darkness for 30 min. Another portion ofsieves 4 Å was added, and a solution of glycosyl chloride (350 mg, 1.0mmol) in dry dichloromethane (3 ml) was added. The mixture was stirredfor 20 h at room temperature. The resin was filtered and washed withmethanol (4×10 ml), then solvent was evaporated. Chromatography onsilica gel (elution with 5-7% isopropanol in chloroform) yielded 407 mg(70%) of the product 3 as a mixture of anomers (α/β=3.0 as determined by¹H-NMR spectroscopy).

A solution of the product 3 (407 mg, 0.352 mmol) in methanol (30 ml) wassubjected to hydrogenolysis over 400 mg 10% Pd/C for 16 h. Then theresin was filtered off, washed with methanol (4×10 ml) and the productconcentrated in vacuum. The dry residue was acetylated with 2:1pyridine-acetic anhydride mixture (6 ml) at 20° C. for 16 h, thereagents being co-evaporated with toluene. Two chromatography steps onsilica gel (elution with 10% isopropanol in ethyl acetate and with 5-10%methanol in chloroform) resulted in 160 mg (42%) of the product 4 and 39mg (10%) of the product 40.

A solution of 2 M sodium methylate in methanol (200 μl) was added to asolution of the product 4 (160 mg, 0.149 mmol) in dry methanol (4 ml).The solution was evaporated after 1 h, 4 ml water added and the solutionkept for 16 h before being chromatographed on a Dowex-H⁺ column (elutionwith 1 M ammonia). The eluate was evaporated, lyophilized to yield 87.2mg (91%) of the 3-aminopropyltrisaccharide (5).

¹H NMR spectra were recorded on a Bruker BioSpin GmbH spectrometer at303K. Chemical shifts (δ) for characteristic protons are provided in ppmwith the use of HOD (4.750), CHCl₃ (δ 7.270) as reference. Couplingconstants (J) are provide in Hz. The signals in ¹H NMR spectra wereassigned using a technique of spin-spin decoupling (double resonance)and 2D-¹H, ¹H-COSY experiments.

The values of optical rotation were measured on a digital polarimeterPerkin Elmer 341 at 25° C.

Mass spectra were registered on a MALDI-TOF Vision-2000 spectrometerusing dihydroxybenzoic acid as a matrix.

4: ¹H-NMR (700 MHz, CDCl₃): 1.759-1.834 (m, 1H, CH sp); 1.853-1.927 (m,1H, CH sp); 1.972, 1.986, 1.996, 2.046, 2.053, 2.087, 2.106, 2.115,2.130, 2.224 (10 s, 10×3H, COCH₃); 3.222-3.276 (m, 1H, NCH sp);3.544-3.583 (m, 1H, OCH sp); 3.591-3.661 (m, 2H, NCH sp, H-5a); 3.764(dd≈t, 1H, H-4a, J 8.8); 3.787 (dd, 1H, H-3b, J_(3,4) 3.7, J_(2,3) 9.9);3.836 (br. t, 1H, H-5b, J 7.3); 3.882-3.920 (m, 1H, OCH sp); 3.950 (dd,1H, H-6′c, J_(6′,6″)10.6, J_(5,6′)5.2); 4.009 (ddd, 1H, H-2a, J_(1,2)7.9, J_(2,3) 10.0, J_(2,NH) 9.0); 4.076-4.188 (m, 5H, H-6′a, H-6′b,H-6″b, H-5c, H-6″c); 4.415 (d, 1H, H-1a, J_(1,2) 7.9); 4.443 (d, 1H,H-1b, J_(1,2) 7.9); 4.529 (dd, 1H, H-6″a, J_(6′,6″)12.0, J_(5,6″)-2.5);4.548 (ddd, 1H, H-2c, J_(1,2) 3.4, J_(2,3) 11.6, J_(2,NH) 9.4); 4.893(dd, 1H, H-3c, J_(3,4) 3.1, J_(2,3) 11.6); 5.021 (d, 1H, H-1c, J_(1,2)3.4); 5.039-5.075 (m, 2H, H-3a, H-2b); 5.339 (dd≈d, 1H, H-4b, J 2.9);5.359 (dd, 1H, H-4c, J_(3,4) 2.7, J_(4,5) 0.9); 5.810 (d, 1H, NHAc a,J_(2,NH) 9.0); 6.184 (d, 1H, NHAc c, J_(2,NH) 9.4); 7.310-7.413 (m, 1H,NHCOCF₃ sp). R_(f) 0.31 (EtOAc-iPrOH, 10:1). MS, m/z calculated for[C₄₃H₆₀N₃F₃O₂₅]H: 1076.35, found 1076.

4β: ¹H-NMR (700 MHz, CDCl₃): 1.766-1.832 (m, 1H, CH sp); 1.850-1.908 (m,1H, CH sp); 1.923, 1.969, 1.982, 2.059, 2.071, 2.099 (2), 2.120, 2.136,2.148 (10 s, 10×3H, COCH₃); 3.230-3.289 (m, 1H, NCH sp); 3.521 (ddd, 1H,H-2c, J_(1,2)8.2, J_(2,3) 11.2, J_(2,NH) 7.8); 3.548-3.591 (m, 1H, OCHsp); 3.591-3.648 (m, 2H, NCH sp, H-5a); 3.743 (dd≈t, 1H, H-4a, J 8.6);3.795 (br. t, 1H, H-5b, J 6.5); 3.852 (dd, 1H, H-3b, J_(3,4) 3.6,J_(2,3) 9.9); 3.873-3.923 (m, 2H, H-5c, OCH sp); 4.002 (ddd, 1H, H-2a,J_(1,2) 8.0, J_(2,3) 9.5, J_(2,NH) 8.9); 4.039 (dd, 1H, H-6′b, J_(6′,6″)11.6, J_(5,6′), 6.9); 4.087-4.144 (m, 3H, H-6′a, H-6″b, H-6′c); 4.160(dd, 1H, H-6″c, J_(6′,6″)11.2, J_(5,6″)6.0); 4.409, 4.417 (2d≈t, 2×1H,H-1a, H-1b, J 7.6); 4.519 (dd, 1H, H-6″a, J_(6′,6″) 11.8, J_(5,6″)2.5);4.992 (d, 1H, H-1c, J_(1,2) 8.2); 5.043 (dd, 1H, H-3a, J_(3,4) 8.6,J_(2,3) 9.5); 5.066 (dd, 1H, H-2b, J_(1,2) 8.0, J_(2,3)9.8); 5.350(dd≈d, 1H, H-4c, J 3.2); 5.372 (dd≈d, 1H, H-4b, J 3.4); 5.399 (d, 1H,NHAc c, J_(2,NH) 7.8); 5.449 (dd, 1H, H-3c, J_(3,4) 3.4, J_(2,3) 11.3);5.856 (d, 1H, NHAc a, J_(2,NH) 8.9); 7.361-7.466 (m, 1H, NHCOCF₃ sp).R_(f) 0.24 (EtOAc-iPrOH, 10:1). MS, m/z calculated for[C₄₃H₆₀N₃F₃O₂₅]H⁺: 1076.35, found 1076.

5: ¹H-NMR (700 MHz, D₂O): 1.924-2.002 (m, 2H, CH₂ sp); 2.060, 2.064 (2s,2×3H, NCOCH₃); 3.102 (m≈t, 2H, NCH₂ sp, J 6.8); 3.592-3.644 (m, 1H,H-5a); 3.655 (dd, 1H, H-2b, J_(1,2) 7.9, J_(2,3) 9.9); 3.702 (br. dd,1H, H-5b, J_(5,6′)3.8, J_(5,6″)8.2, J_(4,5)≤1); 3.713-3.815 (m, 9H);3.846 (dd, 1H, H-6′a, J_(6′,6″)12.3, J_(5,6′)5.3); 3.984-4.062 (m, 4H,OCH sp, H-6″a, H-4b, H-3c); 4.123 (dd≈d, 1H, H-4c, J 2.9); 4.206 (br. t,1H, H-5c, J 6.3); 4.248 (dd, 1H, H-2c, J_(1,2) 3.6, J_(2,3) 11.0); 4.542(2d≈t, 2H, H-1a, H-1b, J 7.4); 5.100 (d, 1H, H-1c, J_(1,2) 3.5). R_(f)0.55 (MeOH-1M aq. Py.AcOH, 5:1). MS, m/z calculated for [C₂₅H₄₅N₃O₁₆]H⁺:644.28; found 644. [α]_(546 nm)+128 (c 0.3; MeCN—H₂O, 1:1).

5β: 1H-NMR (700 MHz, D₂O): 1.938-1.991 (m, 2H, CH₂ sp); 2.055, 2.062(2s, 2×3H, NCOCH₃); 3.100 (m≈t, 2H, NCH₂ sp, J 6.9); 3.610 (dd, 1H,H-2b, J_(1,2) 7.9, J_(2,3) 9.9); 3.603-3.636 (m, 1H, H-5a); 3.682 (br.dd, 1H, H-5b, J_(5,6′) 4.9, J_(5,6″)7.8, J_(4,5)≤1); 3.693-3.826 (m,11H); 3.842 (dd, 1H, H-6′a, J_(6′,6″)12.1, J_(5,6′)5.2); 3.934-3.972 (m,2H, H-4b, H-2c); 4.012 (dd, 1H, H-6″a, J_(6′,6″), 12.2, J_(5,6′)2.0);4.023-4.057 (m, 1H, OCH sp); 4.175 (dd≈d, 1H, H-4c, J 2.9); 4.478 (d,1H, H-1b, J_(1,2) 7.9); 4.531 (d, 1H, H-1a, J_(1,2) 8.1); 4.638 (d, 1H,H-1c, J_(1,2) 8.4). R_(f) 0.48 (MeOH-1M aq. Py.AcOH, 5:1). MS, m/zcalculated for [C₂₅H₄₅N₃O₁₆]H⁺: 644.28; found 644. [α]_(546 nm)+6 (c0.3; MeCN—H₂O, 1:1).

Preparation of3-aminopropyl-α-D-galactopyranosyl-(1-3)-β-D-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside(Galα1-3Galβ1-4GlcNAc-S₁; Galili-S₁) (9) (SCHEME II)

A mixture of the glycosyl acceptor 2 (500 mg, 0.59 mmol),thiogalactopyranoside 6 (576 mg, 1.18 mmol), NIS (267 mg, 1.18 mmol),anhydrous CH₂Cl₂ (25 ml) and molecular sieves 4 Å (500 mg) was stirredat −45° C. for 30 min under an atmosphere of Ar. A solution of TfOH (21μl, 0.236 mmol) in anhydrous CH₂Cl₂ (0.5 ml) was then added. Thereaction mixture was stirred for 2 h at −45° C. and the temperature wasthen increased to −20° C. over 4 h. The mixture was kept at −20° C.overnight. Then extra amounts of thiogalactopyranoside 6 (144 mg, 0.295mmol), NIS (66 mg, 0.295 mmol) and TfOH (5 μl, 0.06 mmol) were added andthe stirring maintained at −20° C. for 2 h before being allowed toslowly warm up to r.t. (1 h). A saturated aqueous solution of Na₂S₂O₃was then added and the mixture filtered. The filtrate was diluted withCHCl₃ (300 ml), washed with H₂O (2×100 ml), dried by filtration throughcotton wool, and concentrated. Gel filtration on LH-20 (CHCl₃-MeOH)afforded the product3-trifluoroacetamidopropyl-3,4-di-O-acetyl-2,6-di-O-benzyl-α-D-galactopyranosyl-(1→3)-2,4-di-1-acetyl-6-O-benzyl-β-D-galactopyranosyl-(1→4)-2-acetamido-3-O-acetyl-6-O-benzyl-2-deoxy-β-D-glucopyranoside(7) (600 mg, 80%), as a white foam.

¹H NMR (700 MHz, CDCl₃, characteristic signals), 6, ppm: 1.78-1.82 (m,4H, CHCHC, OC(O)CH₃), 1.84-1.90 (m, 1H, CHCHC), 1.91, 1.94, 1.97, 1.98,2.06 (5 s, 5×3H, 4 OC(O)CH₃, NH(O)CH₃), 3.23-3.30 (m, 1H, NCHH),3.59-3.65 (m, 1H, NCHH), 4.05 (m, 1H, H-21), 4.33 (d, 1H, J_(1,2) 7.55,H-1^(I)), 4.40 (d, 1H, J 12.04, PhCHH), 4.42 (d, 1H, J_(1,2) 8.07,H-1^(II)), 4.45 (d, 1H, J 11.92, PhCHH), 4.48 (d, 1H, J 12.00, PhCHH),4.50 (d, 1H, J 12.00, PhCHH), 4.52 (d, 1H, J 12.04, PhCHH), 4.54 (d, 1H,J 12.00, PhCHH), 4.57 (d, 1H, J 12.00, PhCHH), 4.64 (d, 1H, J 11.92,PhCHH), 4.99 (dd≈t, 1H, J 8.24, H-2^(II)), 5.08-5.13 (m, 2H, H-31,H-3^(III)), 5.23 (d, 1H, J_(1,2) 3.31, H-1^(III)), 5.46 (d, 1H, J_(3,4)2.25, H-4_(II)), 5.54 (d, 1H, J_(3,4) 3.11, H-4^(III)), 7.20-7.40 (m,20H, ArH); 7.49-7.54 (m, 1H, NHC(O)CF₃). R_(f) 0.4 (PhCH₃—AcOEt, 1:2).

The product 7 (252 mg, 0.198 mmol) was deacetylated according to Zemplen(8 h, 40° C.), neutralized with AcOH and concentrated. The TLC(CH₃Cl-MeOH, 10:1) analysis of the obtained product showed two spots:the main spot with R_(f) 0.45, and another one on the start line(ninhydrin positive spot) that was an indication of partial loss oftrifluoroacetyl. Therefore, the product was N-trifluoroacetylated bytreatment with CF₃COOMe (0.1 ml) and Et₃N (0.01 ml) in MeOH (10 ml) for1 h, concentrated and subjected to column chromatography on silica gel(CHCl₃-MeOH, 15:1) to afford the product 8 as a white foam (163 mg,77%), R_(f) 0.45 (CH₃Cl-MeOH, 10:1). The product 8 was subjected tohydrogenolysis (200 mg Pd/C, 10 ml MeOH, 2 h), filtered,N-defluoroacetylated (5% Et₃N/H₂O, 3 h) and concentrated.Cation-exchange chromatography on Dowex 50X4-400 (H′) (elution with 5%aqueous ammonia) gave the product 9 (90 mg, 98%) as a white foam.

1H NMR (D₂O, characteristic signals), 6, ppm: 1.94-1.98 (m, 2H, CCH₂C),2.07 (s, 3H, NHC(O)CH₃), 3.11 (m, J 6.92, 2H, NCH₂), 4.54 and 4.56 (2d,2H, J_(1,2) 8.06, J_(1,2) 7.87, H-1^(I) and H-1^(II)), 5.16 (d, 1H,J_(1,2) 3.87, H-1^(III)). R_(f) 0.3 (EtOH-BuOH-Py-H₂O-AcOH;100:10:10:10:3).

Preparation of 3-aminopropylα-D-galactopyranosyl-(1→4)-β-D-galactopyranosyl-(1→4)-β-D-glucopyranoside(21) and 2-aminoethylα-D-galactopyranosyl-(1→4)-β-D-galactopyranosyl-(1→4)-β-D-glucopyranoside(28) (Galα1-4Galβ1-4Glc-S₁; Gb₃-S₁)

The title primary aminoalkyl variants of Gb₃-S₁ were prepared accordingSCHEME III, SCHEME IV and SCHEME V.

Preparation of(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-α-D-glucopyranosyltrichloroacetimidate (10)

Trichloroacetonitrile (12.1 ml, 121 mmol) and DBU (0.45 ml, 3 mmol) wereadded to a solution of 10a (7.68 g, 12.1 mmol) in dry dichloromethane(150 ml) at −5° C. The reaction mixture was stirred at −5° C. for 3.5 hand concentrated in vacuo.

Flash chromatography (2:1 to 1:2 (0.1% Et₃N) toluene-ethyl acetate) ofthe residue provided 10 (6.01 g, 63.9%) as a light yellow foam, R_(f)0.55 (2:1 toluene-acetone).

¹H NMR, CDCl₃: 1.95-2.2 (7s, 21H, 7Ac), 4.49 (d, 1H, J_(1,2)=8.07,H-1b), 4.91 (dd, 1H, J_(3,2)=10.3, J_(3,4)=2.8, H-3b), 5.05 (dd, 1H,J_(2,1)=3.5, J_(2,3)=9.3, H-2a), 5.12 (dd, 1H, J_(2,1)=8.07,J_(2,3)=10.3, H-2b), 5.32 (d, 1H, J_(4,3)=³, J_(4,5)<1, H-4b), 5.52 (t,1H, J_(3,2)=J_(3,4)=9.29, H-3a), 6.48 (d, 1H, J_(1,2)=3.5, H-1a), 8.64(s, 1H, HN═CCCl₃).

Preparation of3-chloropropyl-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-β-D-glucopyranoside(11)

A mixture of 2.94 g (3.8 mmol) of trichloroacetimidate 10, 0.66 ml (7.5mmol) 3-chloropropanol, 50 ml dichloromethane, and 3 g of molecularsieves MS 4 Å was cooled to −5° C. An 8% solution of BF₃.Et₂O (0.4 mmol)in anhydrous dichloromethane was added drop wise with stirring.

After 30 min, the reaction mixture was filtered, diluted with chloroform(500 ml), and washed with water, saturated sodium hydrocarbonatesolution, and water to pH 7. The washed reaction mixture was dried byfiltration through a cotton layer and concentrated in vacuo.

Column chromatography on Silica gel (elution with 2.5:1 (v/v)toluene-ethyl acetate) resulted in 1.75 g (65%) of lactose derivative(11) as white foam. R_(f) 0.54 (2:1 toluene-acetone), R_(f) 0.50 (4:2:1hexane-chloroform-isopropanol), [α]_(D)−4° (c 1.0, CHCl₃), m/z 712.2(M⁺).

¹H NMR, CDCl₃: 1.95 (br. s, 5H, Ac, —CH₂—), 2.0-2.2 (6s, 18H, 6Ac), 3.52(m, 2H, —CH₂Cl), 3.63 (m, 1H, H-5a), 3.68 (m, 1H, OCHH—), 3.79 (t, 1H,J=9.3, H-4a), 3.88 (m, 1H, H-5b), 3.93-3.98 (m, 1H, OCHH—), 4.05-4.15(m, 3H, H-6a′, H-6b, H-6b′), 4.45 (d, 2H, H-1a, H-1b, J_(2,1)=7.83) 4.47(m, 1H, H-6a), 4.89 (dd, 1H, J_(2,3)=9.3, J_(2,1)=7.82, H-2a), 4.96 (dd,1H, J_(3,2)=10.5, J_(3,4)=3.42, H-3b), 5.11 (dd, 1H, J_(2,3)=10.5,J_(2,1)=7.83, H-2b), 5.21 (t, 1H, J=9.3, H-3a), 5.35 (dd, 1H,J_(4,3)=3.42, J_(4,5)<1).

Preparation of 3-azidopropyl(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-β-D-glucopyranoside(12)

A mixture of 2.15 g (3 mmol) of trichloropropylglycoside 11, 0.59 g (9mmol) NaN₃, and 30 ml DMSO was maintained at 80° C. with stirring for 20h. The mixture was then diluted with chloroform (500 ml), washed withwater (4×100 ml), dried by filtration through a cotton layer, andconcentrated in vacuo.

Column chromatography on Silica gel (elution with 8:2:1hexane-chloroform-isopropanol) resulted in 1.96 g (91%) of glycoside(12) as a white foam, R_(f) 0.54 (2:1 (v/v) toluene-acetone), R_(f) 0.50(4:2:1 (v/v/v) hexane-chloroform-isopropanol), [α]_(D) −5.4° (c 1.0,CHCl₃), m/z 718.8 (M⁺).

¹H NMR, CDCl₃: 1.85 (m, 2H, —CH₂—), 1.98-2.2 (7s, 21H, 7Ac), 3.36 (m,2H, —CH₂N₃), 3.61 (m, 2H, H-5a, OCHH—CH₂—), 3.8 (t, 1H,J_(3,4)=J_(4,5=9.29), H-4a), 3.85-3.94 (m, 2H, OCHH—CH₂; H-5b),4.05-4.17 (m, 3H, H-6a, H-6a′, H-6b), 4.49 (d, 1H, J_(1,2)=8.07, H-1a),4.5 (m, 1H, H-6b′), 4.51 (d, 1H, J_(1,2=8.07), H-1b), 4.9 (dd, 1H,J_(2,1)=8.07, J_(2,3)=9.29, H-2a), 4.97 (dd, 1H, J_(3,2)=10.27,J_(3,4)=3, H-3b), 5.12 (dd, 1H, J_(2,1)=8.07, J_(2,3)=10.27, H-2b), 5.2(t, 1H, J_(3,2)=J_(3,4)=9.29, H-3a), 5.36 (dd, 1H, J_(4,3)=3,J_(4,5)<1).

Preparation of 3-azidopropyl(4,6-O-benzylidene-β-D-galactopyranosyl)-(1→4)-β-D-glucopyranoside (13)

The lactoside 12 (1.74 g, 2.4 mmol) was deacetylated according toZemplen and co-evaporated with toluene (2×30 ml). The residue wastreated with α,α-dimethoxytoluene (0.65 ml, 3.6 mmol) andp-toluenesulfonic acid (50 mg, to pH 3) in DMF (20 ml) for 3 h. Thereaction mixture was then quenched with pyridine, concentrated, andco-evaporated with o-xylene.

Column chromatography on Silica gel (elution with 9:1 (v/v)chloroform-isopropanol) and recrystalization (chloroform-methanol)resulted in 0.756 mg (62%) of benzylidene derivative (13). R_(f) 0.6(5:1 chloroform-isopropanol), [α]_(D)−25.7° (c 1.0, methanol), m/z 513.4(M⁺).

¹H NMR, CD₃OD: 2.06 (m, 2H, —CH₂—), 3.45 (dd, 1H, J_(2,1)=J_(2,3)-=9,H-2a), 3.61 (m, 1H, H-5a), 3.64 (m, 2H, —CH₂N₃), 3.74-3.9 (m, 6H, OCHH—;H-3a, H-4a; H-2b, H-3b, H-5b), 4.08-4.18 (m, 3H, H-6, H-6a′, OCHH—),4.34-4.44 (m, 3H, H-6b, H-6b′, H-4b), 4.5 (d, 1H, J_(1,2)=7.9, H-1a),4.68 (d, 1H, J_(1,2)=8, H-1b), 5.82 (s, 1H, CHPh), 7.55-7.72 (m, 5H,CHPh).

Preparation of 3-azidopropyl(4,6-O-benzylidene-3-O-benzyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside(14)

Sodium hydride in mineral oil (290 mg, 12 mmol) was slowly added in 4 to5 portions to a solution of 13 (726 mg, 1.5 mmol) in DMF (15 ml) at 0°C. with stirring. After 1 h, the ice bath was removed and benzyl bromidewas added drop wise. The mixture was stirred overnight. 10 ml ofmethanol was then added. After 1 h, the mixture was diluted withchloroform (500 ml), and washed with water (3×200 ml), dried byfiltration through a cotton layer, concentrated, and co-evaporated invacuo with o-xylene.

Column chromatography on Silica gel (elution with 10:1 toluene-ethylacetate) resulted in 1.24 g (87%) of lactose derivative 14 as whitefoam, R_(f) 0.56 (5:3 (v/v) hexane-ethyl acetate), [α]_(D)+10.8° (c 1.0,CHCl₃), m/z 963.8 (M⁺).

¹H NMR, CDCl₃: 1.85 (m, 2H, —CH₂—), 2.91 (m, 1H, H-5b), 3.33 (m, 1H,H-5a), 3.34-3.42 (m, 4H, H-2a, H-3b, —CH₂N₃), 3.55-3.62 (m, 2H, OCHH—;H-3a), 3.73 (dd, 1H, J_(2,1)=8, J_(2,3)=10, H-2b), 3.92-3.97 (m, 2H,H-4a, OCHH—), 4.0 (br. d, 1H, J_(4,3)=3.6, H-4b), 4.34 (d, 1H,J_(1,2=7.9), H-1a), 4.42 (d, 1H, J_(1,2)=8, H-1b), 5.43 (s, 1H, CH(Bd),7.14-7.50 (m, 30H, Ph).

Preparation of 3-azidopropyl(2,3,6-O-tri-O-benzyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside(15)

Hydrogen chloride in diethyl ether was added to a mixture of 14 (1.24 g,1.3 mmol), sodium cyanoborohydride (0.57 g, 9.1 mmol), and freshlyactivated molecular sieves MS 3 Å (33 g) in anhydrous THE (20 ml) untilthe evolution of gas ceased.

The mixture was stirred for 2 h, diluted with chloroform (300 ml),washed with water, saturated sodium hydrocarbonate solution, and waterto pH 7. The washed mixture was dried by filtration through a cottonlayer and concentrated in vacuo.

Column chromatography on Silica gel (elution with 20:1 to 7:3 (v/v)toluene-ethyl acetate) resulted in 0.91 g (65%) of lactose derivative 15as a white foam, R_(f) 0.42 (9:1 (v/v) toluene-acetone), [α]_(D)+17.8°(c 1.0, CHCl₃), m/z 965.8 (M⁺).

¹H NMR, CDCl₃: 1.85 (m, 2H, —CH₂—), 2.39 (d, 1H, J=2.2, OH), 4.04 (br.s, 1H, H-4b), 4.34 (d, 1H, J_(1,2)=7.9, H-1a), 4.42 (d, 1H, J_(1,2)=8,H-1b), 7.14-7.50 (m, 30H, Ph).

¹H NMR of acetylated analytical probe 15a, CDCl₃: 1.85 (m, 2H, —CH₂—),4.34 (d, 1H, J_(1,2)=7.9, H-1a), 4.42 (d, 1H, J_(1,2)=8, H-1b), 5.5 (br.d, 1H, J_(4,3)=3.43, H-4b), 7.14-7.50 (m, 30H, Ph).

Preparation of 3-trifluoroacetamidopropyl(2,3,6-O-tri-O-benzyl-β-D-galactopyranosyl)-(1-4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside(16)

A mixture of derivative 15 (0.914 g, 0.94 mmol), triphenylphosphine (0.5g, 1.9 mmol) and THE (10 ml) was stirred for 0.5 h, 100 μl of wateradded, and the mixture stirred overnight. The reaction mixture was thenconcentrated and co-evaporated with methanol. The residue was dissolvedin methanol (15 ml) and triethylamine (30 μl) and methyltrifluoroacetate (0.48 ml, 4.7 mmol) added. The solution was held for 30min and then concentrated.

Column chromatography on Silica gel (elution with 5:1 to 1:1 (v/v)hexane-acetone) resulted in 0.87 g (84%) of lactose derivative 16 aswhite foam, R_(f) 0.49 (9:1 (v/v) hexane-acetone), [α]_(D)+17° (c 1.0,CHCl₃), m/z 1060.1 (M⁺+Na).

¹H NMR, CDCl₃: 1.88 (m, 2H, —CH₂—), 2.40 (br. s, 1H, OH), 4.05 (br. s,1H, H-4b), 4.36 (d, 1H, J_(1,2)=7.8, H-1a), 4.40 (d, 1H, J_(1,2)=7.6,H-1b), 7.10-7.35 (m, 30H, Ph).

Preparation of 2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyltrichloroacetimidate (18)

A mixture of galactose derivative 17 (2 g, 3.65 mmol),trichloroacetonitrile (1.75 ml, 17.55 mmol), anhydrous potassiumcarbonate (2 g, 14.6 mmol), and dichloromethane (4 ml) was stirred for22 h at room temperature under argon. The mixture was then filteredthrough a Celite layer and concentrated in vacuo. Column chromatographyon Silica gel (elution with 4:1 (v/v) hexane-ethyl acetate (1% Et₃N)resulted in 1.5 g (60%) of 18 as white foam, R_(f) 0.47 (7:3 (v/v)hexane-ethyl acetate containing 1% Et₃N) and 0.46 g (0.6 mmol, 23%) ofthe starting derivative 17, R_(f) 0.27 (7:3 (v/v) hexane-ethyl acetatecontaining 1% Et₃N).

¹H NMR (CDCl₃): 3.60-3.70 (m, 3H, H-3, H-6, H-6′), 3.75 (t, 1H,J_(5,6)=6.30, H-5), 3.98 (d, 1H, J_(4,3)=2.19, H-4), 4.08 (dd, 1H,J_(2,3)=9.73, J_(2,1)=7.95, H-2), 4.42 and 4.47 (ABq, 2H, J=12.00,PhCH₂), 4.63 and 4.95 (ABq, 2H, J=11.51, PhCH₂), 4.72 (s, 2H, PhCH₂),4.80 and 4.90 (ABq, 2H, J=10.95, PhCH₂), 5.74 (d, 1H, J_(1,2)=7.95,H-1), 7.22-7.35 (m, 20H, ArH), 8.62 (s, 1H, NH).

Preparation of 3-trifluoroacetamidopropyl(2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl)-(1→4)-(2,3,6-tri-O-benzyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside(19)

A mixture of lactose derivative 16 (158 mg, 0.153 mmol),trichlroroacetimidate 18 (120 mg, 0.175 mmol), molecular sieves MS 4 Å(0.5 g), and dichloromethane (5 ml) was stirred for 30 min at roomtemperature under argon. 0.1 ml of a 1% (v/v) solution of trimethylsilyltrifluoromethanesulfonate in dichloromethane was then added. After 2 h,another 50 mg (0.073 mmol) trichlroroacetimidate 18 and 30 μl of a 1%(v/v) solution of trimethylsilyl trifluoromethanesulfonate indichloromethane were added. The reaction mixture was stirred overnightat +4° C., quenched with triethylamine (5 μl), filtered, andconcentrated in vacuo.

Column chromatography on Silica gel (elution with 12:1 to 1:1 (v/v)toluene-ethyl acetate) resulted in 170 mg (72%) of trisaccharide 19;R_(f) 0.56 (4:1 (v/v) toluene-ethyl acetate); [α]_(D) +30.8° (c 1.0,CHCl₃).

¹H NMR, CDCl₃: 1.78-1.89 (m, 2H, —CH₂—), 4.34 (d, 1H, J_(1,2)=7.8,H-1a), 4.43 (d, 1H, J_(1,2)=7.4, H-1b), 5.06 (d, 1H, J_(1,2)=3.0, H-1c),7.14-7.48 (m, 50H, Ph).

Preparation of 3-trifluoroacetamidopropyl(2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl)-(1→4)-(2,3,6-tri-O-acetyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-β-D-glucopyranoside(20)

The catalyst 10% Pd/C (10 mg) was added to a solution of the protectedoligosaccharide 19 (73 mg, 0.047 mmol) in methanol (7 ml), the mixturedegassed, and the flask filled with hydrogen. The reaction mixture wasstirred for 1 h, filtered off from the catalyst through a Celite layer,and concentrated in vacuo. The dry residue was dissolved in pyridine (2ml), acetic anhydride (1 ml) added, and the mixture held for 3 h. Thesolvents were then evaporated and residue co-evaporated with toluene(4×2 ml). Column chromatography on Silica gel (elution with 2:1hexane-acetone) resulted in 43.5 mg (90%) of trisaccharide 20 as a whitefoam, R_(f) 0.52 (2:1 hexane-acetone), [α]_(D)+30.4° (c 1.0, CHCl₃).

¹H NMR, CDCl₃: 1.87 (2H, m, CH₂); 1.99, 2.05, 2.05, 2.06, 2.07, 2.07,2.09, 2.09, 2.12, and 2.14 (10×3H, 10 s, 10 Ac); 3.37 and 3.52 (2×1H, 2m, 2 CHN); 3.63 (1H, ddd, J_(4,5)=9.8, J_(5,6)=4.9, J_(5,6)=2.0, H-5a);3.72 (1H, m, OCH); 3.77 (1H, ddd≈br. T, J_(4,5)<1, J_(5,6)=6.8,J_(5,6)=6.1, H-5b); 3.79 (1H, dd, J_(3,4)=9.3, J_(4,5)=9.8, H-4a); 3.87(1H, m, OCH); 4.02 (1H, dd≈br. d, J_(3,4)=2.5, J_(4,5)<1, H-4b); 4.09(1H, dd, J_(5,6)=4.9, J_(6,6′)=12.0, H-6a); 4.12 (1H, dd, J_(5,6)=5.6,J_(6,6′)=10.8, H-6c); 4.14 (1H, dd, J_(5,6)=6.8, J_(6,6′)=11.0, H-6b);4.17 (1H, dd, J_(5,6′)=8.6, J_(6,6′)=10.8, H-6′c); 4.45 (1H, dd,J_(5,6′)=6.1, J_(6,6′)=11.0, H-6′b); 4.49 (1H, ddd □br. T, J_(4,5)<1,J_(5,6)=5.6, J_(5,6′)=8.6, H-5c); 4.50 (1H, d, J_(1,2)=7.8, H-1a); 4.55(1H, d, J_(1,2)=7.8, H-1b); 4.59 (1H, dd, J_(5,6′)=2.0, J_(6,6′)=12.0,H-6′a); 4.76 (1H, dd, J_(2,3)=10.8, J_(3,4)=2.5, H-3b); 4.86 (1H, dd,J_(1,2)=8.1, J_(2,3)=9.5, H-2a); 4.10 (1H, d, J_(1,2)=3.4, H-1c); 5.12(1H, dd, J_(1,2)=7.8, J_(2,3)=10.8, H-2b); 5.19 (1H, dd, J_(1,2)=3.4,J_(2,3)=11.0, H-2c); 5.22 (1H, dd≈T, J_(2,3)=9.5, J_(3,4)=9.3, H-3a);5.40 (1H, dd, J_(2,3)=11.0, J_(3,4)=3.4, H-3c); 5.59 (1H, dd≈br. d,J_(3,4)=3.4, J_(4,5)<1, H-4c); 7.09 (1H, m, NHCOCF₃).

Preparation of 3-aminopropylα-D-galactopyranosyl-(1→4)-β-D-galactopyranosyl-(1→4)-β-D-glucopyranoside(Gb₃-S₁) (21)

Sodium methylate (30 μl of 2 M solution in methanol) was added to asolution of trisaccharide (20) (43 mg, 0.042 mmol) in anhydrous methanol(3 ml) and held for 2 h. The solution was then concentrated in vacuo,water (3 ml) added, and the mixture held for 3 h. The mixture was thenapplied to a column (10×50 mm) with Dowex 50X4-400 (H⁺) cation exchangeresin.

The target compound was eluted with 1 M aqueous ammonia and the eluantconcentrated in vacuo. Lyophilization from water provided trisaccharide21 (23 mg, quant.) as a colorless powder. R_(f) 0.3 (100:10:10:10:2(v/v/v/v/v) ethanol-n-butanol-pyridine-water-acetic acid), [α]_(D)+42°(c 1; water), m/z 584.9 (M⁺+Na).

¹H NMR, D₂O: 1.98-2.05 (m, 2H, —CH₂—), 3.17 (m, 2H, —CH₂NH₂), 3.33-3.35(m, 1H, H-2a), 4.36 (m, 1H, H-5c), 4.53 (d, 2H, J=7.8, H-1a, H-1b), 4.97(d, 1H, J_(1,2)=3.67, H-1c).

Preparation of 2-azidoethyl(3,4-di-O-acetyl-2,6-di-O-benzyl-α-D-galactopyranosyl)-(1→4)-(2,3,6-tri-O-benzyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside(25)

To the solution of ethyl3,4-di-O-acetyl-2,6-di-O-benzyl-1-thio-β-D-galactopyranoside (23) (550mg, 1.11 mmol) in dichloromethane (10 ml) was added Br₂ (57 μl, 1.11mmol). The mixture was held for 20 min at room temperature, thenconcentrated in vacuo at room temperature and co-evaporated withanhydrous benzene (3×30 ml). The crude3,4-di-O-acetyl-2,6-di-O-benzyl-α-D-galactopyranosylbromide (24) wasused for glycosylation without purification.

The mixture of lactose derivative 22 (Sun et al (2006)) (500 mg, 0.525mmol), 1,1,3,3-tetramethylurea (300 μl), molecular sieves MS 4 Å (1 g),and dichloromethane (25 ml) was stirred for 30 min at room temperature.Silver trifluoromethanesulfonate (285 mg, 1.11 mmol), molecular sievesMS 4 Å (0.5 g), and the freshly prepared galactopyranosylbromide (24) indichloromethane (15 ml) were then added. The reaction mixture wasstirred overnight, filtered, and concentrated in vacuo.

Column chromatography on Silica gel (elution with 3:1 to 1:1 (v/v)hexane-ethyl acetate) resulted in 570 mg (79%) of trisaccharide 25,R_(f) 0.25 (2:1 (v/v) hexane-ethyl acetate); [α]_(D)+32° (c 0.8, CHCl₃)

¹H NMR, CDCl₃: 1.88, 1.94 (2s, 2Ac), 3.00 (dd, 1H, J_(5,6)=4.9,J_(6′,6″)=8.4, H-6a), 3.19 (dd, J_(1,2)=8.5, J_(2,3)=8.9, H-2a),3.30-3.36 (m, 2H, —CHHN₃, H-6′a), 3.38-3.47 (m, 4H, H-5a, H-5b, H-2b,H-6b), 3.48-3.54 (m, 1H, —CHHN₃), 3.61 (dd, 1H, J_(2,3)=8.9,J_(3,4)=9.2, H-3a), 3.69-3.75 (m, 3H, H-6′b, H-6c, —OCHH—), 3.85 (dd,1H, J_(5,6)=4.6, J_(6,6′)=11.0, H-6c), 3.89 (dd, 1H, J_(1,2)=3.4,J_(2,3)=10.8, H-2c), 3.95 (dd, 1H, J_(3,4)=9.2, J_(4,5)=9.5, H-4a),4.0-4.1 (m, 4H, —OCHH—, H-4b, CH₂Ph), 4.25, 4.29, 4.32, 4.39 (4 d, 4×1H,J_(AB)=12, 4 —CHPh), 4.43 (d, 1H, J_(1,2)=7.6, H-1), 4.48 (d, 1H,J_(1,2)=7.6, H-1), 4.54-4.62 (m, 5H, 4 —CHPh, H-5c), 4.71-4.84 (m, 4H, 4—CHPh), 4.89, 4.91, and 5.09 (3 d, 3×1H, 3 4 —CHPh), 5.15 (d, 1H,J_(1,2)=3.0, H-1c), 5.39 (dd, 1H, J_(2,3)=10.8, J_(3,4)=3.4, H-3c), 5.56(dd, 1H, J_(3,4)=3.4, J_(4,5)=0.9, H-4c), 7.14-7.48 (m, 40H, Ph).

Preparation of 2-aminoethylα-D-galactopyranosyl-(1→4)-β-D-galactopyranosyl-(1→4)-)β-D-glucopyranoside(Gb₃-S₁) (28)

Sodium methylate (100 μl of 2 M solution in methanol) was added to asuspension of trisaccharide (25) (500 mg, 0.363 mmol) in anhydrousmethanol (50 ml). The mixture was stirred overnight at room temperature,quenched with acetic acid, and concentrated in vacuo.

Column chromatography on Silica gel (elution with 2:1 to 1:1 (v/v)hexane-ethyl acetate) resulted in 470 mg of trisaccharide (26), R_(f)0.5 (1:1 (v/v) hexane-ethyl acetate), [α]_(D)+36° (c 0.5, CHCl₃).

To a solution of trisaccharide (26) and Boc₂O ((150 mg, 0.91 mmol) inanhydrous methanol (50 ml) was added the catalyst 10% Pd/C (500 mg). Themixture was degassed and the flask filled with hydrogen. The reactionmixture was stirred for 3 h, filtered off from the Pd/C, andconcentrated in vacuo.

Column chromatography on Silica gel (elution with 6:5:1 (v/v/v)chloroform-ethanol-water) resulted in 160 mg (68%) of trisaccharide 27R_(f) 0.3 (6:5:1 (v/v/v) dichloromethane-ethanol-water). ¹H NMR, D₂O:1.45 (s, 9H, (CH₃)₃COCO—), 4.53 (d, 1H, J_(1,2)=7.8, H-1b), 4.58 (d, 1H,J_(1,2)=7.4, H-1b), 4.98 (d, 1H, J_(1,2)=3.0, H-1c).

The trisaccharide 27 was then treated with 95% CF₃COOH (5 ml, 10 min).Upon completion, the mixture was concentrated in vacuo, co-evaporatedwith toluene, and applied to a column (10×100 mm) of Dowex 50X4-400 (H′)cation exchange resin. The target compound was eluted with 1 M aqueousammonia and the eluant was concentrated in vacuo. Lyophilization fromwater provided trisaccharide 28 (135, quant.) as a colorless powder.R_(f) 0.35 (100:10:10:10:2 (v/v/v/v/v)ethanol-n-butanol-pyridine-water-acetic acid), [α]_(D)+25° (c 0.2;water).

¹H NMR, D₂O:: 3.32 (m, 2H, —CH₂NH₂), 3.40-3.45 (m, 1H, H-2a), 3.63 (dd,1H, J_(1,2)=7.9, J_(2,3)=10.3, H-2b), 3.66-3.78 (m, 5H, H-5a, H-3a,H-4a, H-6c, H-6′c), 3.8 (dd, 1H, J_(3,4)=3.1, J_(3,2)=10.3, H-3b), 3.84(m, 2H, J_(5,6)=4.4, J_(5,6′)=7.9, H-5b), 3.88-3.92 (m, 3H, H-2c, H-6b,—OCHH—), 3.96 (dd, 1H, J_(3,4)=3.3, J_(3,2)=10.3, H-3c), 3.98-4.03 (m,2H, H-6a, H-6′b), 4.06 (dd, 1H, J_(5,6)=2.2, J_(6,6′)-=12.3, H-6′a),4.08 (dd, 1H, J_(3,4)=3.3, J_(4,5)=0.9, H-4c), 4.09 (d, 1H, J_(3,4)=3.1,H-4b), 4.17-4.21 (m, 1H, —OCHH—), 4.41 (m, 1H, H-5c), 4.56 (d, 1H,J=7.9, H-1b), 4.60 (d, 1H, J=8.1, H-1a), 5.00 (d, 1H, J_(1,2)=3.9,H-1c).

The preparation of the primary amino propyl glycosidesGalNAcα1-3(Fucα1-2)Galβ-O(CH₂)₃NH₂ (A_(tri)-S₁) andGalα1-3(Fucα1-2)Galβ-O(CH₂)₃NH₂ (B_(tri)-S₁) is described in thepublication of Korchagina and Bovin (1992). The preparation of theprimary amino propyl glycosidesGalα1-3(Fucα1-2)Galβ1-3GlcNAcβ-O(CH₂)₃NH₂ (B_(tetra) (Type 1)-S₁),Galα1-3 (Fucα1-2) Galβ1-3GlcNAcα-O(CH₂)₃NH₂ (B_(tetra)(Type 3)-S₁) andGalα1-3 (Fucα1-2) Galβ1-3GalNAcβ-O(CH₂)₃NH₂ (B_(tetra) (Type 4)-S₁) isdescribed in the publication of Korchagina et al (2009). The preparationof the primary amino propyl glycosides Xylα1-3Glcβ-O(CH₂)₃NH₂ andXylα1-3Xylα1-3Glcβ-O(CH₂)₃NH₂ is described in the publication of Krylovet al (2007). The preparation of the primary amino propyl glycosidesNeu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ-O(CH₂)₃NH₂ (sLe^(x)-S₁) andNeu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ-O(CH₂)₃NH₂ (sLe^(x)-S₁) is describedin the publication of Nifant'ev et al (1996). The preparation of theprimary amino propyl glycosidesGalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ-O(CH₂)₃NH₂ (A_(tetra)(Type 2)-S₁),Galα1-3(Fucα1-2)Galβ1-4GlcNAcβ-O(CH₂)₃NH₂ (B_(tetra) (Type 2)-S₁)Fucα1-2Galβ1-4GlcNAcβ-O(CH₂)₃NH₂ (H_(tri) (Type 2)-S₁),Galβ1-4(Fucα1-3)GlcNAcβ-O(CH₂)₃NH₂ (Le^(x)-S₁), Fucα1-2Galβ1-4(Fucα1-3)GlcNAcβ-O(CH₂)₃NH₂ (Le^(y)-S₁), Galα1-3Galβ1-4GlcNAcβ-O(CH₂)₃NH₂(B_(tri) (Type 2)-S₁) and Galα1-4Galβ1-4GlcNAcβ-O(CH₂)₃NH₂ (P₁-S₁) isdescribed in the publication of Pazynina et al (2002). The preparationof the primary amino propyl glycosides Neu5Acα2-3Galβ-O(CH₂)₃NH₂,Neu5Acα2-3Galβ1-4GlcNAcβ-O(CH₂)₃NH₂ (3′ SLN-S₁),Neu5Acα2-3Galβ1-4(6-HSO₃)GlcNAcβ-O(CH₂)₃NH₂ (6-Su-3′SLN-S₁),Neu5Acα2-3Galβ1-3GalNAcα-O(CH₂)₃NH₂ (SiaTF-S₁),Neu5Acα2-3Galβ1-3(6-HSO₃)GalNAcα-O(CH₂)₃NH₂ (6-Su-SiaTF-S₁),Neu5Acα2-3Galβ1-3GlcNAcβ-O(CH₂)₃NH₂ (SiaLe^(c)-S₁),Neu5Acα2-3Galβ1-3(6-HSO₃)GlcNAcβ-O(CH₂)₃NH₂ (6-Su-SiaLe^(c)-S₁),Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ-O(CH₂)₃NH₂ (SiaLe^(a)-S₁),Neu5Acα2-3Galβ1-4 (Fucα1-3) GlcNAcβ-O(CH₂)₃NH₂ (SiaLe^(x)-S₁) isdescribed in the publication of Pazynina et al (2003). The preparationof the primary amino propyl glycosideGalβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-O(CH₂)₃NH₂(trilactosamine-S₁) is described in the publication of Pazynina et al(2008). The preparation of the primary amino propyl glycosidesGalNAcα1-3 (Fucα1-2) Galα1-3GlcNAcβ-O(CH₂)₃NH₂ (A_(tetra) (Type 1)-S₁),GalNAcα1-3(Fucα1-2)Galβ1-3GlcNAcα-O(CH₂)₃NH₂ (A_(tetra)(Type 3)-S₁) andGalNAcα1-3(Fucα1-2)Galβ1-3GalNAcβ-O(CH₂)₃NH₂ (A_(tetra)(Type 4)-S₁) isdescribed in the publication of Ryzhov et al (2012). The preparation ofa number of primary aminoethyl mono- and disaccharides is described inthe publication of Sardzik et al (2010). The preparation of the primaryamino propyl glycosides Neu5Ac-α-(2-6′)-Galβ1-4GlcNAcβ-O(CH₂)₃NH₂(Neu5Ac-α-(2-6′)-lactosamine-S₁) andNeu5Gc-α-(2-6′)-Galβ1-4GlcNAcβ-O(CH₂)₃NH₂(Neu5Gc-α-(2-6′)-lactosamine-S₁) is described in the publication ofSherman et al (2001). The preparation of primary amino propyl glycosidesof linear β-(1-3)-D-glucooloigosaccharides containing from 3 to 13monosaccharide units is described in the publication of Yashunsky et al(2016).

Several fluorescent compounds are available commercially and may be usedin the preparation of synthetic molecule constructs. For example,fluorescein isothiocyanate (FITC) may be conjugated with a diamine suchas 1,5-diaminopentyl (cadaverine) and dipyrrometheneboron difluoride(BODIPY™) may be conjugated with an alkionyl diamine such as propionlyethylenediamine (BODIPY™ FL EDA). Synthetic molecule constructs where Fis one of the following fluorophores (represented as neutrally chargedprotonated species) may thereby be prepared:

Preparation of S₂-L

Preparation of activated1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (SuNO-Ad-DOPE;S₂-L) (31) (SCHEME VIA)

To a solution of bis(N-hydroxysuccinimidyl) adipate (29) (70 mg, 205μmol) in dry N,N-dimethylformamide (1.5 ml),1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE)(30) (40μmol) in chloroform (1.5 ml) was added, followed by triethylamine (7μl). The mixture was kept for 2 h at room temperature, then neutralizedwith acetic acid and partially concentrated under vacuum. Columnchromatography (Sephadex LH-20, 1:1 chloroform-methanol, 0.2% aceticacid) of the residue yielded the activated lipid 31 (37 mg, 95%) as acolorless syrup. TLC (chloroform-methanol-water, 6:3:0.5): R_(f)=0.5

31: ¹H NMR (CDCl₃/CD₃OD, 2:1) 5.5 (m, 4H, 2×(—CH═CH—), 5.39 (m, 1H,—OCH₂—CHO—CH₂O—), 4.58 (dd, 1H, J=3.67, J=11.98, —CCOOHCH—CHO—CH₂O—),4.34 (dd, 1H, J=6.61, J=11.98, —CCOOHCH—CHO—CH₂O—), 4.26 (m, 2H,PO—CH₂—CH₂—NH₂), 4.18 (m, 2H, —CH₂—OP), 3.62 (m, 2H, PO-CH₂—CH₂—NH₂),3.00 (s, 4H, ONSuc), 2.8 (m, 2H, —CH₂—CO (Ad), 2.50 (m, 4H, 2×(—CH₂—CO),2.42 (m, 2H, —CH₂—CO (Ad), 2.17 (m, 8H, 2×(—CH₂—CH═CH—CH₂—), 1.93 (m,4H, COCH₂CH₂CH₂CH₂CO), 1.78 (m, 4H, 2×(COCH₂CH₂—), 1,43, 1.47 (2 bs,40H, 20 CH₂), 1.04 (m, 6H, 2 CH₃).

Preparation of activated1,2-O-distereoyl-sn-glycero-3-phosphatidylethanolamine (SuNO-Ad-DSPE;S₂-L) (33) (SCHEME VIB)

To a solution of bis(N-hydroxysuccinimidyl) adipate (29) (70 mg, 205μmol) in dry N,N-dimethylformamide (1.5 ml) was added1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE)(32) (40μmol) in chloroform (1.5 ml) followed by triethylamine (7 μl). Themixture was kept for 2 h at room temperature, then neutralized withacetic acid and partially concentrated in vacuo. Column chromatography(Sephadex LH-20, 1:1 chloroform-methanol, 0.2% acetic acid) of theresidue yielded the activated lipid 33 (37 mg, 95%) as a colorlesssyrup; TLC (chloroform-methanol-water, 6:3:0.5): R_(f)=0.55.

33: ¹H NMR (CDCl₃/CD₃OD, 2:1), 5.39 (m, 1H, —OCH₂—CHO—CH₂O—), 4.53 (dd,1H, J=3.42, J=11.98, —CCOOHCH—CHO—CH₂O—), 4.33 (dd, 1H, J=6.87, J=11.98,—CCOOHCH—CHO—CH₂O—), 4.23 (m, 2H, PO—CH₂—CH₂—NH₂), 4.15 (m, 2H,—CH₂—OP), 3.61 (m, 2H, PO—CH₂—CH₂—NH₂), 3.00 (s, 4H, ONSuc), 2.81 (m,2H, —CH₂—CO (Ad), 2.48 (m, 4H, 2×(—CH₂—CO), 2.42 (m, 2H, —CH₂—CO (Ad),1.93 (m, 4H, COCH₂CH₂CH₂CH₂CO), 1.78 (m, 4H, 2×(COCH₂CH₂—), 1,43, 1.47(2 bs, 40H, 20CH₂), 1.04 (m, 6H, 2CH₃).

Preparation of F-S₁-S₂-L

Condensing activated DOPE (or DSPE) with aminopropylglycoside.

To a solution of activated DOPE (or DSPE) (A-L) (33 μmol) inN,N-dimethylformamide (1 ml) 30 μmol of Sug-S₁—NH₂ (F—S₁—NH₂) and 5 μlof triethylamine were added. For example, the Sug may be either theaminopropyl glycoside (F—S₁—NH₂) of either GalNAcα1-3(Fucα1-2)Galβtrisaccharide (A-glycotope) (F) or Galα1-3(Fucα1-2)Galβ trisaccharide(B-glycotope) (F).

The mixture was stirred for 2 h at room temperature. Columnchromatography (Sephadex LH-20 in 1:1 chloroform-methanol followed bysilica gel in ethyl acetate-isopropanol-water, 4:3:1 (v/v/v) of themixture typically yielded 85-90% of the synthetic molecule construct,for example, A_(tri)-sp-Ad-DOPE (I) or B_(tri)-sp-Ad-DOPE (VI).

¹H NMR (CDCl₃/CD₃OD, 1:1), δ:

A_(tri)-sp-Ad-DOPE (I)—5.5 (m, 4H, 2×(—CH═CH—), 5.43-5.37 (m, 2H, H-1(GalNHAc) and —OCH₂—CHO—CH₂O—), 5.32 (d, 1H, H-1, J=3.5 H-1 Fuc), 2.50(m, 4H, 2×(—CH₂—CO), 2.40 (m, 4H, COCH₂CH₂CH₂CH₂CO), 2.20 (m, 8H,2×(—CH₂—CH═CH—CH₂—), 2.1 (s, 3H, NHAc), 1.92 (m, 2H, O—CH₂CH₂CH₂—NH),1.8 (m, 8H, COCH₂CH₂CH₂CH₂CO and 2×(COCH₂CH₂—), 1,43, 1.47 (2 bs, 40H,20 CH₂), 1.40 (d, 3H, J=6.6, CH₃ Fuc), 1.05 (m, 6H, 2 CH₃).

A_(tri)-spsp₁-Ad-DOPE (II)—5.5 (m, 4H, 2×(—CH═CH—), 5.43-5.37 [m, 2H,H-1 (GalNHAc) and —OCH₂—CHO—CH₂O—], 5.32 (d, 1H, H-1, J=3.6 H-1 Fuc),2.50 (m, 4H, 2×(—CH₂—CO), 2.40-2.32 (m, 6H, COCH₂CH₂CH₂CH₂CO and COCH₂—(sp₁), 2.18 [m, 8H, 2×(—CH₂—CH═CH—CH₂—)], 2.1 (s, 3H, NHAc), 1.95 (m,2H, O—CH₂CH₂CH₂—NH), 1.8 [m, 10H, COCH₂CH₂CH₂CH₂CO, 2×(COCH₂CH₂— . . .), —COCH₂CH₂ (CH₂)₃NH—], 1.68 (m, 2H, CO(CH₂)₃CH₂CH₂NH—), 1,43, 1.47 (2bs, 42H, 22 CH₂), 1.37 (d, 3H, J=5.6, CH₃ Fuc), 1.05 (m, 6H, 2 CH₃).

A_(tri)-sp-Ad-DSPE (III)—5.42-5.38 (m, 2H, H-1 (GalNHAc) and—OCH₂—CHO—CH₂O—), 5.31 (d, 1H, H-1, J=3.5 H-1 Fuc), 2.48 [m, 4H,2×(—CH₂—CO)], 2.42 (m, 4H, COCH₂CH₂CH₂CH₂CO), 2.18 (s, 3H, NHAc), 1.95(m, 2H, O—CH₂CH₂CH₂—NH), 1.8 [m, 8H, COCH₂CH₂CH₂CH₂CO and2×(COCH₂CH₂—)], 1,43, 1.47 (2 bs, 56H, 28 CH₂), 1.38 (d, 3H, J=6.6, CH₃Fuc), 1.05 (m, 6H, 2 CH₃).

B_(tri)-sp-Ad-DOPE (VI)—5.5 (m, 4H, 2×(—CH═CH—), 5.42-5.38 [m, 2H, H-1(Gal) and —OCH₂—CHO—CH₂O—], 5.31 (d, 1H, H-1, J=3.7, H-1 Fuc), 2.48 [m,4H, 2×(—CH₂—CO)], 2.39 (m, 4H, COCH₂CH₂CH₂CH₂CO), 2.18 [m, 8H,2×(—CH₂—CH═CH—CH₂—)], 1.93 (m, 2H, O—CH₂CH₂CH₂—NH), 1.8 [m, 8H,COCH₂CH₂CH₂CH₂CO and 2×(COCH₂CH₂—)], 1,43, 1.47 (2 bs, 40H, 20 CH₂),1.36 (d, 3H, J=6.6, CH₃ Fuc), 1.05 (m, 6H, 2 CH₃).

H_(tri)-sp-Ad-DOPE (VII)—5.5 [m, 4H, 2×(—CH═CH—)], 5.4 (m, 1H,—OCH₂—CHO—CH₂O—), 5.35 (d, 1H, H-1, J=3.2, H-1 Fuc), 4.65, 4.54 (2d,J=7.4, J=8.6, H-1 Gal, H-1 GlcNHAc), 4.46 (dd, 1H J=3.18, J=12,—CCOOHCH—CHO—CH₂O—), 4.38-4.28 (m, 2H, H-5 Fuc, CCOOHCH—CHO—CH₂O—), 2.48[m, 4H, 2×(—CH₂—CO)], 2.40 (m, 4H, COCH₂CH₂CH₂CH₂CO), 2.18 [m, 8H,2×(—CH₂—CH═CH—CH₂—)], 2.08 (s, 3H,NHAc), 1.92 (m, 2H, O—CH₂CH₂CH₂—NH),1.82-1.72 [m, 8H, COCH₂CH₂CH₂CH₂CO and 2×(COCH₂CH₂—)], 1,48, 1.45 (2 bs,40H, 20 CH₂), 1.39 (d, 3H, J=6.5, CH₃ Fuc), 1.05 (m, 6H, 2 CH₃).

H_(di)-sp-Ad-DOPE (VIII)—5.49 (m, 4H, 2×(—CH═CH—), 5.37 (m, 1H,—OCH₂—CHO—CH₂O—), 5.24 (d, 1H, H-1, J=2.95, H-1 Fuc), 4.46 (d, J=7.34,H-1 Gal), 2.48 [m, 4H, 2×(—CH₂—CO)], 2.42-2.35 (m, 4H,COCH₂CH₂CH₂CH₂CO), 2.17 [m, 8H, 2×(—CH₂—CH═CH—CH₂—)], 1.95 (m, 2H,O—CH₂CH₂CH₂—NH), 1.81-1.74 [m, 8H, COCH₂CH₂CH₂CH₂CO and 2×(COCH₂CH₂—)],1,45, 1.41 (2 bs, 40H, 20 CH₂), 1.39 (d, 3H, J=6.5, CH₃ Fuc), 1.03 (m,6H, 2 CH₃).

Galβ-sp-Ad-DOPE (IX)—5.51 [m, 4H, 2×(—CH═CH—)], 5.4 (m, 1H,—OCH₂—CHO—CH₂O—), 4.61 (dd, 1H J=3.18, J=12, —CCOOHCH—CHO—CH₂O—), 4.41(d, J=7.8, H-1 Gal), 4.37 (dd, 1H, J=6.6, J=12, —CCOOHCH—CHO—CH₂O—),2.50 [m, 4H, 2×(—CH₂—CO)], 2.40 (m, 4H, COCH₂CH₂CH₂CH₂CO), 2.20 [m, 8H,2×(—CH₂—CH═CH—CH₂—)], 1.97 (m, 2H, O—CH₂CH₂CH₂—NH), 1.82-1.72 [m, 8H,COCH₂CH₂CH₂CH₂CO and 2×(COCH₂CH₂—)], 1,48, 1.45 (2 bs, 40H, 20 CH₂),1.05 (m, 6H, 2 CH₃).

Preparation of GalNAcα1-3Galβ1-4GlcNAc-Ad-DOPE (SCHEME VII)

To a solution of the product 8 (33 μmol) in N,N-dimethylformamide (1ml), 30 μmol of the 3-aminopropyltrisaccharide 5 and 5 μl oftriethylamine (Et₃N) were added. The mixture was stirred for 2 h at roomtemperature. Column chromatography on silica gel (CH₂Cl₂-EtOH-H₂O;6:5:1) provided an 81% yield of the construct 9.

9: ¹H NMR (700 MHz, CDCl₃-CD₃OD, 1:1 v/v, selected), 8, ppm: 1.05 (t,6H, J 7.05, 2 CH₃), 1.39-1.55 (m, 40H, 20 CH₂), 1.75-1.84 (m, 8H,COCH₂CH₂CH₂CH₂CO and 2×COCH₂CH₂—), 1.84-1.96 (m, 2H, O—CH₂CH₂CH₂—NH),2.15-2.22 (m, 14H, 2×(—CH₂—CH═CH—CH₂—), 2×NHC(O)CH₃), 2.34-2.46 (m, 4H,2×-CH₂—CO), 2.36-2.44 (m, 4H, 2×-CH₂—CO), 3.29-3.34 (m, 1H,—CH₂—CHH—NH), 4.17-4.20 (m, 2H, —CHO—CH₂OP—), 4.34-4.39 (m, 2H,—CH₂OPO—CH₂—CH₂), 4.57 (d, 1H, J_(1,2) 8.39, H-1′), 4.50 (dd, 1H, J3.78, J 10.82, —C(O)OCHHCHOCH₂O—), 4.58-4.61 (m, 2H, H-1^(II),C(O)OCHHCHOCH₂O—), 5.15 (d, 1H, J_(1,2) 3.76, H-1^(III)), 5.38-5.42 (m,1H, —OCH₂—CHO—CH₂O—), 5.47-5.53 (m, 4H, 2×-CH═CH—). R_(f) 0.5(CH₂Cl₂-EtOH-H₂O; 6:5:1).

Preparation of Gb₃-S₁(sp3)-Ad-DOPE and Gb₃-S₂(sp2)-Ad-DOPE (SCHEME VIII)

To a solution of activated DOPE (19) (10.5 μmol) in dichloromethane (300μl) was added (12) or (16) (10 μmol) in DMF (0.5 ml) and thentriethylamine (3 μl). The mixture was kept for 2 h at room temperature.Gel filtration on Sephadex LH-20 (1:1 (v/v) chloroform-methanol) of themixture yielded (I) or (III) (90-95%).

Gb₃-sp3-Ad-DOPE (I) was determined to have a molecular weight (MW) of1415.7 and ¹H NMR (CDCl₃/CD₃OD, 2:1), δ: 5.5 (m, 4H, 2×(—CH═CH—),5.43-5.39 (m, 1H, —OCH₂—CHO—CH₂O—), 5.13 (d, 1H, J=3.6, H-1 Gal),4.61-4.58 (m, 2H; J=7.1, H-1 (Gal); J=3.7, J=12.1, —CCOOHCH—CHO—CH₂O—),4.46 (d, J=7.9, H-1 Gal), 2.53-2.48 (m, 4H, 2×(—CH₂—CO), 2.42-2.37 (m,4H, COCH₂CH₂CH₂CH₂CO), 2.21-2.16 (m, 8H, 2×(—CH₂—CH═CH—CH₂—), 2.00-1.95(m, 2H, O—CH₂CH₂CH₂—NH), 1.78 (m, 8H, COCH₂CH₂CH₂CH₂CO and2×(COCH₂CH₂—), 1.50, 1.47 (2 bs, 40H, 20 CH₂), 1.05 (m, 6H, 2 CH₃) (FIG.1).

Gb₃-sp2-Ad-DOPE (III) was determined to have a molecular weight (MW) of1415.7 and ¹H NMR (CDCl₃/CD₃OD, 2:1), δ: 5.5 (m, 4H, 2×(—CH═CH—),5.43-5.39 (m, 1H, —OCH₂—CHO—CH₂O—), 5.13 (d, 1H, J=3.6, H-1 Gal),4.61-4.58 (m, 2H; J=7.1, H-1 (Gal); J=3.7, J=12.1, —CCOOHCH—CHO—CH₂—),4.46 (d, J=7.9, H-1 Gal), 2.53-2.48 (m, 4H, 2×(—CH₂—CO), 2.42-2.37 (m,4H, COCH₂CH₂CH₂CH₂CO), 2.21-2.16 (m, 8H, 2×(—CH₂—CH═CH—CH₂—), 1.78 (m,8H, COCH₂CH₂CH₂CH₂CO and 2×(COCH₂CH₂—), 1.50, 1.47 (2 bs, 40H, 20 CH₂),1.05 (m, 6H, 2 CH₃).

Preparation of Galili-sp-Ad-DOPE (SCHEME IX)

The reactions were performed with the use of commercial reagents (Acros,Aldrich, and Fluka); anhydrous solvents were purified according to thestandard procedures. Column chromatography was performed on Silica gel60 0.040-0.063 mm (Merck), gel filtration was carried out on SephadexLH-20 (GE Healthcare) columns. Solvents were removed in vacuum at 30-40°C. Thin layer chromatography (TLC) was performed on Silica gel 60 F₂₅₄aluminium-backed plates (Merck). Spots of compounds were visualized bydipping a TLC plate into aqueous solution of H₃PO₄ (8%) and subsequentheating (>150° C.).

¹H NMR spectra were recorded on a Bruker BioSpin GmbH (700 MHz)spectrometer at 30° C.; chemical shifts (6, ppm) were referred to thepeak of internal D₂O (δ 4.750), CDCl₃ (δ 7.270), or CD₃OD (δ 3.500);coupling constants (J) were measured in Hz. Signals of ¹H NMR. Symbolsof monosaccharide residues in NMR spectra for saccharides: I—β-GlcNAc(reducing end), II—β-Gal, III—α-Gal. MALDI TOF MS spectra were recordedon Bruker Daltonics Ultraflex MALDI TOF/TOF Mass Spectrometer (Germany).

To a solution of 3-aminopropyl4-O-[3-O—(α-D-galactopyranosyl)-β-D-galactopyranosyl]-2-acetamido-2-deoxy-β-D-glucopyranoside(33) (Mendeleev Communications, 2002, (143-145) or Tetrahedron, 61,(2005), 4313-4321), 52 mg, 0.086 mmol) in dry DMF (2 mL) was added 15 μLof Et₃N followed by a solution of DOPE-Ad-ONSu (31) (U.S. Pat. No.8,013,131 B2, 100.6 mg, 1.00 mmol) in CH₂Cl₂ (2 mL). The reaction wasstirred for 2 hours at room temperature followed by sequential columnchromatography (the first on Sephadex LH-20, and the second on silicagel eluting with CH₂Cl₂-EtOH-H₂O; 6:5:1) to provide the constructdesignated Galili-sp-Ad-DOPE (34) (105.6 mg, 84%).

R_(f) 0.5 (CH₂Cl₂-EtOH-H₂O; 6:5:1). ¹H NMR (700 MHz, CDCl₃-CD₃OD 1:1,30° C.), δ, ppm, selected: 5.45-5.54 (m, 4H, 2×-CH═CH—), 5.34-5.43 (m,1H, —OCH₂—CHO—CH₂O—), 5.18 (d, 1H, J_(1,2) 2.52, H-1^(III)), 4.61 (d,1H, J_(1,2) 7.57, H-4^(II)), 4.60 (dd, 1H, J 2.87, J 12.00,C(O)OCHHCHOCH₂O—), 4.56 (d, 1H, J_(1,2) 8.39, H-1^(I)), 4.36 (dd, 1H, J6.8, J 12.00, —C(O)OCHHCHOCH₂O—), 4.19 (d, 1H, J_(3,4) 2.48, H-4^(II)),4.13-4.18 (m, 2H, —CHO—CH₂OP—), 3.52-3.62 (m, 3H, PO—CH₂—CH₂—NH,—CH₂—CHH—NH), 3.29-3.35 (m, 1H, —CH₂—CHH—NH), 2.45-2.52 (m, 4H,2×-CH₂—CO), 2.36-2.45 (m, 4H, 2×-CH₂—CO), 2.14-2.22 (m, 11H,2×(—CH₂—CH═CH—CH₂—), NHC(O)CH₃), 1.85-1.96 (m, 2H, O—CH₂CH₂CH₂—NH),1.73-1.84 (m, 8H, COCH₂CH₂CH₂CH₂CO and 2×(COCH₂CH₂—), 1.36-1.55 (m, 40H,20 CH₂), 1.05 (t, 6H, J 6.98, 2 CH). C₇₀H₁₂₆N₃O₂₆P; MALDI MS: m/z 1480(M Na+H); 1496 (MK+H); 1502 (MNa+Na), 1518 (M Na+K)

Condensation of DOPE-A with 5-((5-aminopentyl)thioureidyl) fluorescein(fluorescein cadaverine)

To a solution of activated DOPE (L-A) (5 mg, 5.2 μmol) inN,N-dimethylformamide (0.5 ml) 3 mg (4.6 μmol) of fluorescein cadaverinedihydrobromide salt and 5 μl of triethylamine were added. The mixturewas kept for 2 h at room temperature, then 10 μl of 3% aq. NH₃ wereadded and the mixture was kept at room temperature for 1 h.

Column chromatography (Sephadex LH-20, 1:1 chloroform-methanol, followedby silica gel, ethyl acetate-isopropanol-water, 6:3:1) of the mixtureyielded 4.2 mg (67%) KODE-fluorescein (I), R_(f) 0.5 (ethylacetate-isopropanol-water, 6:3:1).

¹H NMR (CDCl₃/CD₃OD, 1:1), δ:

KODE-fluorescein (I)—8.38 (bs, 1H, aromatic proton of fluorescein), 8.15(dd, 1H, J=1.7, J=8.3, aromatic proton of fluorescein) 7.30 (d, 1H,J=8.3, aromatic proton of fluorescein), 6.87 (m, 4H, aromatic protons offluorescein), 6.72 (dd, 2H, J=2.4, J=8.8, aromatic protons offluorescein), 5.50 (m, 4H, 2×(—CH═CH—), 5.38 (m, 1H, —OCH₂—CHO—CH₂O—),4.58 (dd, 1H, J=6.6, J_(gem)=11.8, HHC—O—C(O)—), 4.34 (dd, 1H, J=3.2,J_(gem)-11.8, HHC—O—C(O)—), 4.14 (m, 2H, —OCH—CH₂—O—P—) (4.1 (m, 2H,—P—O—CH₂—CH₂—NH—) 3.80 (m, 2H, N—CH₂(CH₂)₃—CH₂NH—C═S) 3.39 and 3.58 (2m, 2×2H, N— CH₂—CH₂—O—P— and N—CH₂—(CH₂)₃—CH₂NH—C═S)2.48 (m, 4H,2×(—CH₂—CO), 2.39 (m, 4H, COCH₂CH₂CH₂CH₂CO), 2.19 (m, 8H,2×(—CH₂—CH═CH—CH₂—), 1.84 (m, 2H, CH₂-fluorescein cadaverine), 1.8 (m,10H, COCH₂CH₂CH₂CH₂CO, 2×(COCH₂CH₂—, and CH₂—fluorescein cadaverine),1.62 (m, 2H, CH₂— fluorescein cadaverine) 1,42, 1.46 (2 bs, 40H, 20CH₂), 1.05 (m, 6H, 2 CH₃).

Condensation of DOPE-A with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylenediamine, hydrochloride (BODIPYFL EDA)

To a solution of 15 mg (15.5 μmol) activated DOPE in CH₂Cl₂ (0.5 ml), 5mg (13.5 μmol) of BODIPY FL EDA in N,N-dimethylformamide (0.3 ml) and 5μl of triethylamine were added. The mixture was kept for 2 h at roomtemperature.

Column chromatography (Sephadex LH-20, 1:1 chloroform-methanol) of themixture yielded 14.2 mg (75%) KODE-BODIPY (I), Et₃N-salt; MW 1289.6,R_(f) 0.3 (ethyl acetate-isopropanol-water, 6:3:1).

¹H NMR (CDCl₃/CD₃OD, 1:1): b 7.40 (s, 1H, aromatic proton of BODIPY),7.12 (d, 1H, J=3.8 aromatic proton of BODIPY), 6.47 (d, 1H, J=3.8aromatic proton of BODIPY), 6.32 (s, 1H, aromatic protons of BODIPY),5.50 (m, 4H, 2×(—CH═CH—), 5.38 (m, 1H, —OCH₂—CHO—CH₂O—), 4.58 (dd, 111,J=3.2, J_(gem)=11.8, HHC—O—C(O)—), 4.33 (dd, 1H, J=6.6, J_(gem)=11.8,HHC—O—C(O)—), 4.16 (t, 2H, J=5.6, P—O—CH₂—CH₂—NH—), 4.1 (m, 2H,—OCH—CH₂—O—P—), 3.60 (t, 2H, P—O—CH₂—CH₂—NH—), 3.46, 3.42 and 2.8 (3 m,4H, 2H, 2H, —CH₂—CH₂—C(O)NH(CH₂)₂—NH of BODIPY), 2.70 (s, 3H, CH₃ ofBODIPY), 2.48 (m, 4H, 2×(—CH₂—CO), 2.45 (s, 3H, CH₃ of BODIPY), 2.37 (m,4H, COCH₂CH₂CH₂CH₂CO), 2.19 (m, 8H, 2×(—CH₂—CH═CH—CH₂—), 1.8 (m, 8H,COCH₂CH₂CH₂CH₂CO, 2×(COCH₂CH₂—)), 1.46, 1.43 (2 bs, 40H, 20 CH₂), 1.05(m, 6H, 2 CH₃); 3.31 (q, 6H, J=7.4, 3×CH₂ of Et₃N), 1.50 (t, 9H, J=7.4,3×CH₃ of Et₃N).

Example 2—Solubility of Synthetic Glycolipids

For use in the transformation of cells the first criterion thatsynthetic glycolipids must satisfy is that they are soluble in aqueoussolvents, e.g. phosphate buffered saline. A number of techniques,including heat and/or sonication, were employed initially in order tomaximise the solubility of the synthetic glycolipids tested (Table 21).

The synthetic glycolipid must also be able to insert into the membraneand be recognisable to the appropriate antibody for transformation to bedetected by agglutination. Initial tests on the molecules were toestablish solubility and thus eliminate those molecules that wereunsuitable for use in the transformation of cells.

The results of these initial tests are provided in Table 22.

TABLE 21 The range of synthetic glycolipid molecules tested. DOPE LipidTails: B_(tri)-sp-Ad-DOPE (VI) A_(tri)-sp-Ad-DOPE (I) Galβ-sp-Ad-DOPE(IX) H_(di)-sp-Ad-DOPE (VIII) H_(tri)-sp-Ad-DOPE (VII)A_(tri)-spsp₁-Ad-DOPE (II) B_(tri)-PAA-DOPE (V) Different Lipid Tails:A_(tri)-sp-lipid (IV) A_(tri)-sp-Ad-DSPE (III)

TABLE 22 Solubility of synthetic glycolipids in hot PBS andtransformation ability. Water Detectable Synthetic solubilitytransformation ability A_(tri)-sp-lipid (IV) No No B_(tri)-PAA-DOPE (V)No No B_(tri)-sp-Ad-DOPE (VI) Yes Yes A_(tri)-sp-Ad-DOPE (I) Yes YesGalβ-sp-Ad-DOPE Yes No (IX) H_(di)-sp-Ad-DOPE Yes No (VIII)H_(tri)-sp-Ad-DOPE Yes Yes (VII) A_(tri)-spsp₁-Ad-DOPE Yes Yes (II)A_(tri)-sp-Ad-DSPE Yes Yes (III)

The lack of detectable transformation for Galβ-sp-Ad-DOPE (IX) andH_(di)-sp-Ad-DOPE (VIII) was thought to be due to the inability of theantibody to recognise the glycotope of these synthetic molecules.A_(tri)i-sp-lipid (IV) has a single rather than a diacyl tail and it wasproposed that there was no insertion of this synthetic molecule into themembrane bilayer.

Example 3—Low Temperature Transformation of RBCs by A_(tri)-Sp-Ad-DOPE(I) and B_(tri)-Sp-Ad-DOPE (VI) Synthetic Glycolipids

RBCs are healthier when stored at 4° C., and likewise are believed to behealthier when transformed at 4° C. It was not thought that asignificant rate of insertion of the synthetic glycolipids would occurat 4° C. due to our previous studies (see Comparative Examples) andstudies by others (Schwarzmann, 2000). These studies were performed withnatural glycolipids. Surprisingly these studies did not predict thebehaviour of the synthetic glycolipids of the invention.

Whilst not wishing to be bound by theory, in the studies of Schwarzmannthe low rate of insertion of the natural glycolipids may be due to thephysicochemical properties of the natural glycolipid tail; asphingolipid and a fatty acid.

The diacyl tail of the glycolipid may be important in determining therate of insertion. Certain diacyl tails may retain greater fluidity atlower temperatures. Alternatively, the domain of the plasma membraneinto which the diacyl tail of these glycolipids inserts may retain thisgreater fluidity.

It is known that the sphingolipid tails of natural glycolipidscongregate in rigid domains and these domains may not allow furtherincorporation of glycolipid at low temperatures. Synthetic glycolipidswith cis-desaturated diacyl tails may be favoured for use.

Transformation of RBCs with synthetic glycolipids with different lipidtails was first evaluated (Tables 22 and 24).

TABLE 23 Antisera used to obtain results presented in Tables 24 to 27.Manufacturer Catalogue ref Batch number Expiry date Anti-A Albaclone,SNBTS Experimental Z0010770 12.12.04 BioClone, OCD reagent 01102 —Anti-B Albaclone, SNBTS Experimental Z0110600 27.04.03 BioClone, OCDreagent 01103 —

Transformation of RBCs with synthetic glycolipids A_(tri)-sp-Ad-DOPE (I)and B_(tri)-sp-Ad-DOPE (VI) at 4° C. was then evaluated (Tables 25 to28). These transformations were directed towards the preparation ofcells expressing low levels of A, B or A and B glycotopes (“weak A, Band AB cells”).

For the preparation of weak A and B cells transformation solutions (20μL, A_(tri)-sp-Ad-DOPE (I) at 0.08, 0.05 and 0.03 mg/mL, andB_(tri)-sp-Ad-DOPE (VI) at 0.6, 0.3, 0.15, 0.08, 0.05 and 0.03 mg/mL) in1× PBS were mixed with washed, packed group O RBCs (60 μL).

For the preparation of weak AB cells transformation solutions (20 μL,A_(tri)i-sp-Ad-DOPE (I) at 0.07, 0.06 and 0.05 mg/mL, andB_(tri)-sp-Ad-DOPE (VI) at 0.3, and 0.2 mg/mL) in 1× PBS were combinedin block titre with washed, packed group O RBCs (60 μL). Thecombinations were: A_(tri)-sp-Ad-DOPE (I) at 0.07mg/mL+B_(tri)-sp-Ad-DOPE (VI) at 0.3 mg/mL; A_(tri)-sp-Ad-DOPE (I) at0.07 mg/mL+B_(tri)-sp-Ad-DOPE (VI) at 0.2 mg/mL; A_(tri)-sp-Ad-DOPE (I)at 0.06 mg/mL+B_(tri)-sp-Ad-DOPE (VI) at 0.3 mg/mL; A_(tri)-sp-Ad-DOPE(I) at 0.06 mg/mL+B_(tri)-sp-Ad-DOPE (VI) at 0.2 mg/mL;A_(tri)-sp-Ad-DOPE (I) at 0.05 mg/mL+B_(tri)-sp-Ad-DOPE (VI) at 0.3mg/mL; and A_(tri)-sp-Ad-DOPE (I) 0.05+B_(tri)-sp-Ad-DOPE (VI) 0.2mg/mL.

TABLE 24 Evaluation of insertion of different lipid tails byagglutination with the relevant antisera. Anti- Transformation solution(μg/mL) Molecule sera 1000 500 250 125 100 60 50 40 30 20 10A_(tri)-sp-Ad- Alba w+ w+ 0 0 0  DOPE (I) Bio 2+  1+ w+ 0 0  Alba 4+ 3+2-3+ 2+ Bio  4+*  4+*  3+* 3+ DBA 0 B_(tri)-sp-Ad- Alba  3+ DOPE (VI)Bio  3+ Alba  2+ 2+ 1+ 0 0 Bio  3+ 2+ 1+ 0 0 A_(tri)-spsp₁-Ad- Alba 0 00 0 0  DOPE (III) Bio 0 0 0 0 0  Alba 4+ 3+ 2+ 2+ Bio  4+*  3-4+*  3+*2+ DBA 0 A_(tri)-sp-lipid Alba 0 (IV) Bio 0 A_(tri)-sp-Ad- Alba 0 0 0 00  DSPE (III) Bio 0 0 0 0 0  Alba 2-3+ 2-3+ 2+ 2+ Bio 3+ 2-3+ 2+ 2+ DBA0 *splatter.

Cells and transformation solutions were placed in a V° C. fridge.Pipette mixing was performed at intervals. Cells were removed fortesting at intervals against the relevant antisera and were tested inboth washed and unwashed states (i.e. washed samples had thetransformation solution removed).

After 46 hours Celpresol™ was added to the cells so that the finalcells:non-cells ratio was 3:5 (v/v). The cells continued to be tested atintervals. Testing was discontinued after 10 days because cells turnedbrown.

This discolouration could be attributed to a number of factorsincluding: cells were already 21 days old when transformed; 48 hourtransformation was in PBS not Celpresol™ so cells stressed for thistime; and cells may have been mishandled in transit between thetransforming and testing laboratories. This may be mitigated bytransformation of the cells in Celpresol™ as opposed to PBS.

TABLE 25 Diamed results of weak A RBCs transformed at 4° C. againstanti-A. A_(tri)-sp-Ad-DOPE (I) (mg/mL) Washed unwashed Time 0.08 0.050.03 0.08 0.05 0.03 2 hrs 0  0 0 0  0  0 4 hrs 1+ 0 0 2+ 0  0 6 hrs 2+ 00 2+ 0  0 8 hrs 2+ 0 0 2-3+ 0  0 12 hrs 2-3+ 0 0 3+ 1+ 0 24 hrs 3-4+  1+0 3-4+ 2+ 0 30.5 hr 3-4+  1+ 0 3-4+ 2+ 0 48 hrs 4+  2+ 0 4+ 2+ 0 72 hrs4+  2+ 0 4+ 2-3+ 0 96 hrs 4+ 2-3+ 0 4+ 2-3+ 0 Day 7 3-4+ 2+ 0 Day 103-4+ 2+ 0

TABLE 26 Diamed results of weak B RBCs transformed at 4° C. againstanti-B. B_(tri)-sp-Ad-DOPE (VI) (mg/mL) washed unwashed Time 0.6 0.30.15 0.6 0.3 0.15 2 hrs 0  0 0 0  0  0 4 hrs 0  0 0 1+ 0  0 6 hrs w+ 0 01+ 0  0 8 hrs 2+ 0 0 2+ w+ 0 12 hrs 2+  w+ 0 2-3+ 2+ 0 24 hrs 4+  3+  2+4+ 3+  2+ 30.5 hr 4+ 2-3+ 0 4+ 2-3+  w+ 48 hrs 4+  3+  1+ 4+ 3+  2+ 72hrs 4+  4+  2+ 4+ 4+  2+ 96 hrs 4+ 3-4+ 2-3+ 4+ 3-4+ 2-3+ Day 7 4+ 2-3+0 Day 10 4+ 2+ 0

TABLE 27 Diamed results of weak AB RBCs transformed at 4° C. in blocktitre against anti-A. B_(tri)-sp-Ad- A_(tri)-sp-Ad-DOPE (I) (mg/mL) DOPE(VI) washed unwashed Day (mg/mL) 0.07 0.06 0.05 0.07 0.06 0.05 1 0.3 2+1-2+ w+ 2-3+ 2+ 1+ 0.2 2+ 1-2+ 0  2-3+ 2+ 1+ 5 0.3 2+ 1-2+ 1+ 2-3+ 2+1-2+ 0.2 2+ 1-2+ w+ 2-3+ 2+ 1-2+ 8 0.3 2-3+ 2+ 2+ 0.2 2-3+ 2+ 1-2+

Example 4—Insertion Efficiency of Transformation of RBCs byA_(tri)-Sp-A-DOPE (I) and B_(tri)-Sp-Ad-DOPE (VI) Synthetic Glycolipids

The post-transformation supernatant solutions (from A_(tri)-sp-Ad-DOPE(I) at 0.08 mg/mL, 0.05 mg/mL and 0.03 mg/mL, and B_(tri)-sp-Ad-DOPE(VI) at 0.6 mg/mL, 20 μL) were added neat and in a 1:2 dilution towashed, packed RBCs (60 μL). The tubes were incubated in a 37° C.waterbath for one hour, with mixing taking place every 15 minutes.

TABLE 28 Diamed results of weak AB RBCs transformed at 4° C. in blocktitre against anti-B. B_(tri)-sp-Ad- A_(tri)-sp-Ad-DOPE (I) (mg/mL) DOPE(VI) washed unwashed Day (mg/mL) 0.07 0.06 0.05 0.07 0.06 0.05 1 0.3 3+3+ 2+ 3+ 3+ 2-3+ 0.2 1+ 1-2+ 0  2+ 2+ 1-2+ 5 0.3 2+ 2+ 1+ 2+ 2+ 2+ 0.20  w+ vw 1+ w+ vw 8 0.3 2+ 2+ 2+ 0.2 1+ 1+ 0 

The transformed RBCs were washed 3× with PBS and then suspended inCellstab™ at the appropriate concentration for serology testing.

TABLE 29 Tube serology. Pre-trans conc (mg/mL) Score A_(tri)-sp-Ad-DOPE(I) at 0.08 0 1:2 of A_(tri)-sp-Ad-DOPE (I) at 0.08 0 A_(tri)-sp-Ad-DOPE(I) at 0.05 0 1:2 of A_(tri)-sp-Ad-DOPE (I) at 0.05 0 A_(tri)-sp-Ad-DOPE(I) at 0.03 0 1:2 of A_(tri)-sp-Ad-DOPE (I) at 0.03 0 B_(tri)-sp-Ad-DOPE(VI) at 0.60 vw+ 1:2 of B_(tri)-sp-Ad-DOPE (VI) at 0.60 0

The score given by the post-transformation supernatant solution (fromthe 0.08 mg/mL pre-transformation solution) is not even that of the 0.03mg/mL transformation solution in the first pass (w+). These resultsindicate that >75% of the molecules are inserted into the RBC membraneon the first pass.

In addition, the post-transformation solutions were concentrated 20× andcompared in parallel with the transformation solutions of knownconcentration. Only the post-transformation solutions derived from the0.08 mg/mL A_(tri)-sp-Ad-DOPE (I) and 0.6 mg/mL B_(tri)-sp-Ad-DOPE (VI)solutions were tested.

Post-transformation solutions (20 μL) were dialysed (pore size 500 Da)against de-ionised water for 2 days. The samples were left to dry in afumehood for 10 days. At the end of this time they were transferred intoa rotavapor flask and set on the rotavapor to rotate under vacuum withno heat overnight. Samples were dried in a water bath at 40° C. andwashed over into smaller vessels with chloroform-methanol 2:1 leavingsignificant amounts of dried cellular material. The chloroform-methanol2:1 washings were dried down, washed over again into test-tubes withchloroform-methanol 2:1 and dried down. These samples were redissolvedin 1 mL of 1× PBS and used for transformation experiments. The cellularmaterial in the bottom of the flasks was washed out with water intoanother set of tubes.

The post-transformation solutions (from A_(tri)-sp-Ad-DOPE (I) at 0.08mg/mL and B_(tri)-sp-Ad-DOPE (VI) at 0.6 mg/mL, 20 μL) were added towashed, packed RBCs (60 μL). In parallel, the transformation solutions(A_(tri)-sp-Ad-DOPE (I) at 0.08 mg/mL, 0.05 mg/mL and 0.03 mg/mL, andB_(tri)-sp-Ad-DOPE (VI) at 0.6 mg/mL, 20 μL) were added to washed,packed RBCs (60 μL).

The tubes were incubated in a 37° C. waterbath for one hour, with mixingtaking place every 15 minutes. The transformed RBCs were washed 3× withPBS and then suspended in Cellstab™ at the appropriate concentration forserology testing.

TABLE 30 Diamed serology. Conc (mg/mL) Score A_(tri)-sp-Ad-DOPE (I) at0.08 3+ A_(tri)-sp-Ad-DOPE (I) at 0.05 2+ A_(tri)-sp-Ad-DOPE (I) at 0.031+ From A_(tri)-sp-Ad-DOPE (I) at 0.08 0 B_(tri)-sp-Ad-DOPE (VI) at 0.604+ From B_(tri)-sp-Ad-DOPE (VI) at 0.60 0

These results suggest that there are not enough molecules in thepost-transformation solution, even when concentrated 20×, to be detectedby serology.

Example 5—Transformation of Murine RBCs by H_(tri)-Sp-Ad-DOPE (VII)Synthetic Glycolipid

TABLE 31 Anti-H reagents used for results in Tables 32 and 33. AntiseraManufacturer Batch Anti-H IgM Japanese Red Cross HIRO-75 UEA LorneLaboratories 11549E D.O.E. 06.2004 Bio-UEA EY Labs 201105-2

TABLE 32 Tube Serology. H Antisera UEA Cells IgM T = 0 T = 20 Bio-UEAMouse RBCs (- control) 0 0 0 Mouse RBCs + 0.01 mg/mL 0H_(tri)-sp-Ad-DOPE (VII) Mouse RBCs + 0.05 mg/mL 1+ H_(tri)-sp-Ad-DOPE(VII) Mouse RBCs + 0.1 mg/mL 3+ H_(tri)-sp-Ad-DOPE (VII) Mouse RBCs +0.25 mg/mL 4+ 1+ H_(tri)-sp-Ad-DOPE (VII) Mouse RBCs + 1 mg/mL 2+ 2+H_(tri)-sp-Ad-DOPE (VII) Human O RBCs (+ control) 4+ 1+ 2/3+ 4+

TABLE 33 Diamed. Cells Score Mouse RBCs + 0.01 mg/ml H_(tri)-sp-Ad-DOPE(VII) 0 Mouse RBCs + 0.05 mg/ml H_(tri)-sp-Ad-DOPE (VII) 0 Mouse RBCs +0.1 mg/ml H_(tri)-sp-Ad-DOPE (VII) 2+ Mouse RBCs + 0.25 mg/mlH_(tri)-sp-Ad-DOPE (VII) 3+

Example 6—Transformation of RBCs by Filtered A_(tri)-Sp-Ad-DOPE (I)Synthetic Glycolipid

Some A_(tri)-sp-Ad-DOPE (I) had been sterile-filtered through a 0.2 μmfilter. To investigate whether transformation would be the same withthis product a comparative trial was done.

TABLE 34 Anti-A used for results presented in Table 35. ManufacturerCatalogue ref Batch number Expiry date BioClone, OCD Experimental 01102— reagent

TABLE 35 Column agglutination of A RBCs transformed with varyingconcentrations of sterile-filtered vs unfiltered A_(tri)-sp-Ad-DOPE (I).Concentration Sterile-filtered A_(tri)-sp-Ad- Unfiltered A_(tri)-sp-Ad-(mg/mL) DOPE (I) DOPE (I) 0.2 4+ 4+ 0.1 4+ 3-4+ 0.05 2-3+ 2-3+ 0.01 0 0Control 37° C. 0 Control 25° C. 0

These results show no significant difference between the twopreparations of A_(tri)-sp-Ad-DOPE (I) and suggests that filtrationthrough a 0.2 μM filter did not remove molecules or change thecomposition or properties of the fluid to the point that transformationwas affected.

Example 7—Storage of Transformed Cells

To investigate whether storage at 4° C. or 37° C. changed theagglutination results of A_(tri)-sp-Ad-DOPE (I) and natural A glycolipidtransformed O RBCs, identified as “Syn-A” and “Nat-A” cellsrespectively, were divided in two and suspended to 5% in Cellstab™.

One set of cells was stored at 4° C. and the other set of cells wasstored at 37° C. in a waterbath. Agglutination of the stored transformedcells was assessed (Table 36).

TABLE 36 Syn-A A_(tri)-sp-Ad- Nat-A Time Plat- Temp DOPE (I) at At At(hours) form (° C.) 0.1 mg/mL 1 mg/mL 10 mg/mL Control 0 Tube 3+ 0 1-2+0 20 Column 4 4+ 0 3+ 0 37 4+ 0 3+ 0 44 Column 4 4+ 3+ 0 37 4+ 3+ 0

Example 8—RBC Transformation with A- and B-Antigen Synthetic Glycolipidswith Different Non-Carbohydrate Structures

The water soluble synthetic glycolipids designated A_(tri)-sp-Ad-DOPE(I), A_(tri)-sp₁sp₂-Ad-DOPE (II), A_(tri)-sp-Ad-DSPE (III), andB_(tri)-sp-Ad-DOPE (VI) were prepared according to the method describedin Example 1 with necessary modifications.

Washed packed group O red blood cells (RBCs) (3 parts by volume) and thesynthetic glycolipid solution (1 part by volume, varying concentrations)were added to an eppendorf tube. The tube was incubated in a 37° C.waterbath for one hour, mixing every 15 minutes. The transformed RBCswere washed 3× with PBS and then suspended in Cellstab™ at theappropriate concentration for serology testing.

Tube serology and Diamed gel-card results for RBCs transformed with thedifferent synthetic molecule constructs are provided in Table 38.Results for the stability of the RBCs transformed with the differentsynthetic glycolipids at different concentrations are provided in Tables39 to 44.

TABLE 37 Antisera used for results presented in Tables 38 to 44.Antisera Manufacturer Batch Albaclone anti-A SNBTS Z0010770 - D.O.E12.12.04 Bioclone anti-A Ortho Diagnostics 01102 - D.O.M 16.05.02Albaclone anti-B SNBTS Z0110670 - D.O.E 12.12.04 Bioclone anti-B OrthoDiagnostics 01103 - D.O.M 16.05.02

TABLE 38 Comparison of transformation of RBCs using A-antigen syntheticglycolipids at different concentrations. A Antisera Conc Albacloneanti-A Bioclone anti-A Synthetic mg/mL Tube Diamed Tube DiamedA_(tri)-sp-Ad-DOPE (I) 0.25 n.d. 4+ n.d. 4+ 0.1 n.d. 4+/3+ n.d. 4+/3+0.05 w+ 2+ 2+ 2+ 0.04 w+ n.d. 1+ n.d. 0.03 0 n.d. w+ n.d. 0.02 0 n.d. 0n.d. 0.01 0 0 0 0 A_(tri)-sp-Ad-DSPE (III) 0.25 n.d. 0 n.d. 0 0.1 n.d. 0n.d. 0 0.05 0 0 0 0 0.04 0 n.d. 0 n.d. 0.03 0 n.d. 0 n.d. 0.02 0 n.d. 0n.d. 0.01 0 0 0 0 A_(tri)-sp₁sp₂-Ad-DOPE (II) 0.25 n.d. 4+ n.d. 4+ 0.1n.d. 4+ n.d. 4+/3+ 0.05 0 3+ 0 3+ 0.04 0 n.d. 0 n.d. 0.03 0 n.d. 0 n.d.0.02 0 n.d. 0 n.d. 0.01 0 0 0 0 Incubated control — 0 n.d. 0 n.d. Benchcontrol — 0 n.d. 0 n.d. Abbreviations: n.d. Not determined.

TABLE 39 Stability trial of RBCs transformed with Atri-sp-Ad- DOPE (I)at high concentrations (1 mg/mL, 0.5 mg/mL and 0.25 mg/mL).Agglutination by manual tube serology. Cell Albaclone anti-A Biocloneanti-A storage Concentration of Transformation Solution (mg/mL) Daysolution 1 0.5 0.25 1 0.5 0.25 2 Alsevers 4+ 4+ 4+ 4+^(o) 4+^(o) 4+^(o)Cellstab ™ 4+ 4+ 3+ 4+^(o) 4+^(o) 4+^(o) 10 Alsevers 3+ 2+ 2+ 4+^(o)4+^(o) 3+  Cellstab ™  4+^(o)  3+^(o) 2+ 4+^(o) 4+^(o) 4+^(o) 17Alsevers 4+ 4+ 4+ 4+^(o) 4+^(o) 4+^(o) Cellstab ™ 4+ 4+ 4+ 4+^(o) 4+^(o)4+^(o) 24 Alsevers 4+ 4+ 4+ 4+  4+  4+  Cellstab ™ 4+ 4+ 4+ 4+^(o) 4+ 4+ 

TABLE 40 Stability trial of RBCs transformed with A_(tri)-sp-Ad-DOPE (I)at low concentrations (0.1 mg/mL, 0.05 mg/mL and 0.025 mg/mL).Agglutination by manual tube serology. Cell Albaclone anti-A Biocloneanti-A storage Concentration of Transformation Solution (mg/mL) Daysolution 0.1 0.05 0.025 0.1 0.05 0.025 2 Alsevers 3+/2+ 1+ 1+/w+ 2+2+/1+ 1+ Cellstab ™ 3+/2+ 2+  1+ 3+/2+ 3+/2+ 2+ 8 Alsevers 2+ 1+  w+3+/2+ 2+ 2+ Cellstab ™ 2+ 1+/w+ vw  3+^(o) 2+ 1+ 15 Alsevers 2+ 1+ 0 3+2+ Vw Cellstab ™ 4+ w+ 0 4+ 4+ 1+ 22 Alsevers 2+ 2+ 0 3+ 2+ w+Cellstab ™ 4+ 4+  1+ 4+ 4+ 1+ 44 Alsevers n.d. n.d. n.d. n.d. n.d. n.d.Cellstab ™ 4+ 2+  w+ 4+ 2+ w+

TABLE 41 Stability trial of RBCs transformed with A_(tri)-sp-Ad-DOPE (I)at high concentrations (1 mg/mL, 0.5 mg/mL and 0.25 mg/mL).Agglutination in Diamed gel-cards. Cell Albaclone anti-A Bioclone anti-Astorage Concentration of Transformation Solution (mg/mL) Day solution 10.5 0.25 1 0.5 0.25 2 Alsevers 4+ 4+ 4+ 4+ 4+ 4+ Cellstab ™ 4+ 4+ 4+ 4+4+ 4+ 10 Alsevers 4+ 4+ 4+ 4+ 4+ 4+ Cellstab ™ 4+ 4+ 4+ 4+ 4+ 4+ 17Alsevers 4+ 4+ 4+ 4+ 4+ 4+ Cellstab ™ 4+ 4+ 4+ 4+ 4+ 4+ 24 Alsevers 4+4+ 4+ 4+ 4+ 4+ Cellstab ™ 4+ 4+ 4+ 4+ 4+ 4+ 45 Alsevers 4+ 4+ 4+ 4+ 4+4+ Cellstab ™ 4+ 4+ 4+ 4+ 4+ 4+ 59 Alsevers 4+ 4+ 4+ 4+ Cellstab ™ 4+ 4+4+ 4+ 4+ 4+ 73 Alsevers Cellstab ™ 4+ 4+ 4+ 4+ 4+ 4+ 88 AlseversCellstab ™ 4+ 4+ 4+ 4+ 4+ 4+

TABLE 42 Stability trial of RBCs transformed with A_(tri)-sp-Ad-DOPE (I)at low concentrations (0.1 mg/mL, 0.05 mg/mL and 0.025 mg/mL).Agglutination in Diamed gel-cards. Where there were insufficient cellsfor testing, blank spaces have been left. Cell Albaclone anti-A Biocloneanti-A storage Concentration of Transformation Solution (mg/mL) Daysolution 0.1 0.05 0.025 0.1 0.05 0.025 2 Alsevers 4+ 2+ 0 4+ 3+ 1+Cellstab ™ 4+ 2+ 0 4+ 3+ 1+ 8 Alsevers 4+ 3+ 0 4+ 4+ 1+ Cellstab ™ 4+ 3+0 4+ 4+ 1+ 15 Alsevers 4+ 2+ 0 4+ 3+/2+ 1+ Cellstab ™ 4+ 4+ 0 4+ 4+ 1+22 Alsevers 4+ 3+/2+ 0 4+ 3+ w+ Cellstab ™ 4+ 4+ 0 4+ 4+ 1+ 29 Alsevers4+ 2+ 0 4+ 3+ w+ Cellstab ™ 4+ 3+ 0 4+ 4+ 2+ 43 Alsevers 4+ 3+  w+ 4+ 4+2+ Cellstab ™ 4+ 4+/3+ 0 4+ 4+ 1+ 50 Alsevers 4+ 3+  w+ 4+ 4+ 2+Cellstab ™ 4+ 3+ 0 4+ 4+ 1+ 57 Alsevers 4+ 3+/2+ 4+ 4+ Cellstab ™ 4+ 3+0 4+ 3+ w+ 63 Alsevers Cellstab ™ 4+/3+ 2+ 0 4+ 3+ 0  71 AlseversCellstab ™ 4+/3+ 2+ 0 4+ 3+ 0  86 Alsevers Cellstab ™ 4+/3+ 2+ 0 4+ 3+0 

TABLE 43 Stability trial of RBCs transformed with B_(tri)-sp-Ad-DOPE(VI) at high concentrations (1 mg/mL, 0.5 mg/mL and 0.25 mg/mL).Agglutination by manual tube serology (^(o)—splatter). Cell Albacloneanti-B Bioclone anti-B storage Concentration of Transformation Solution(mg/mL) Day solution 1 0.5 0.25 1 0.5 0.25 2 Alsevers 3+ 3+ 2+ 2+ 1+ 1+Cellstab ™ 3+ 2+ 2+ 2+ 2+ 1+ 9 Alsevers 4+ 4+ 2+ 4+ 3+ 2+ Cellstab ™ 4+4+ 3+ 4+ 4+ 2+ 16 Alsevers 4+ 4+ 3+ 4+ 4+ 2+ Cellstab ™ 4+ 4+ 2+ 4+ 4+2+ 23 Alsevers 4+ 4+ 3+ 4+ 4+ 3+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 3+ 30Alsevers 3+ 3+ 2+ 2+ 2+ 2+ Cellstab ™ 4+ 3+ 2+  3+^(o)  3+^(o) 2+ 37Alsevers 3+ 2+ 1+ 3+ 2+ 1+ Cellstab ™ 3+ 3+ 2+/1+  4+^(o) 3+ 1+ 44Alsevers 4+ 3+ 1+ 3+ 3+ w+ Cellstab ™ 4+ 4+ n.d. 4+ 4+ ^(‡) 51 Alsevers3+ 3+ 2+ 4+ 3+ 2+ Cellstab ™ 4+ 4+ n.d. 4+ 4+ 2+

TABLE 44 Stability trial of RBCs transformed with B_(tri)-sp-Ad-DOPE(VI) at high concentrations (1 mg/mL, 0.5 mg/mL and 0.25 mg/mL).Agglutination in Diamed gel-cards. Where there were insufficient cellsfor testing, blank spaces have been left. Cell Albaclone anti-B Biocloneanti-B storage Concentration of Transformation Solution (mg/mL) Daysolution 1 0.5 0.25 1 0.5 0.25 2 Alsevers 4+ 4+ 2+ 4+ 4+ 2+ Cellstab ™4+ 4+ 2+ 4+ 4+ 2+ 9 Alsevers 4+ 4+ 2+ 4+ 4+ 2+ Cellstab ™ 4+ 4+ 3+ 4+ 4+3+ 16 Alsevers 4+ 4+ 2+ 4+ 4+ 1+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 3+ 23Alsevers 4+ 4+ 3+ 4+ 4+ 3+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 3+ 30 Alsevers 4+4+ 3+ 4+ 4+ 3+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 3+ 37 Alsevers 4+ 4+ 3+ 4+ 4+3+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 3+ 44 Alsevers 4+ 4+ 2+ 4+ 4+ 3+ Cellstab ™4+ 4+ 3+ 4+ 4+ 4+/3+ 51 Alsevers 4+ 4+ 2+ 4+ 4+ 3+ Cellstab ™ 4+ 4+ 3+4+ 4+ 3+ 58 Alsevers 4+ 1+ 4+ 2+ Cellstab ™ 4+ 4+ 2+ 4+ 4+ 2+ 72Alsevers 4+ 2+ 4+ 3+ Cellstab ™ 4+ 4+ 3+/2+ 4+ 4+ 3+ 87 AlseversCellstab ™ 4+ 4+/3+ 1+ 4+ 4+/3+ 2+/1+ 116 Alsevers Cellstab ™ 4+ 3+ 0 4+ 4+/3+ 1+

Example 9—Red Blood Cell Transformation with R-Antigen SyntheticGlycolipids

The water-soluble synthetic glycolipids designated H_(tri)-sp-Ad-DOPE(VII), H_(di)-sp-Ad-DOPE (VIII) and Galβ-sp-Ad-DOPE (IX) were preparedaccording to the method described in Example 1 with necessarymodifications.

Washed packed mouse RBCs (3 parts by volume) and the syntheticglycolipid solutions (1 part by volume of varying concentrations) wereadded to an eppendorf tube. The tube was incubated in a 37° C. waterbathfor one hour, mixing every 15 minutes. The transformed RBCs were washed3× with PBS and then suspended in Cellstab™ at the appropriateconcentration for serology testing.

Tube serology and Diamed gel-card results for RBCs transformed with thedifferent synthetic glycolipids are presented in Table 46. The resultsshow that three sugars (H_(tri)) are required for detection by anti-HIgM, at least by the reagent used.

TABLE 45 Antisera used for results presented in Table 46. AntiseraManufacturer Batch Anti-H IgM Japanese Red Cross HIRO-75 UEA LorneLaboratories 11549E D.O.E. 06.2004 Bio-UEA EY Labs 201105-2

TABLE 46 Comparison of transformation of RBCs using H-antigen syntheticglycolipids with different glycotopes made to different concentrations(n.d.—not determined). H Antisera UEA Conc IgM Tube Tube Bio-UEASynthetic mg/mL Tube Diamed T0 T20 Tube H_(tri)-sp- 1 n.d. n.d. 2+ n.d.2+ Ad-DOPE 0.25  4+ 3+ n.d. n.d. 1+ (VII) 0.1  3+ 2+ n.d. n.d. n.d. 0.05 1+ 0  n.d. n.d. n.d. 0.01 0 0  n.d. n.d. n.d. H_(di)-sp- 0.25 0 n.d.n.d. n.d. n.d. Ad-DOPE 0.1 0 n.d. n.d. n.d. n.d. (VIII) 0.05 0 n.d. n.d.n.d. n.d. 0.01 0 n.d. n.d. n.d. n.d. Galβ-sp- 0.25 0 n.d. n.d. n.d. n.d.Ad-DOPE 0.1 0 n.d. n.d. n.d. n.d. (IX) 0.05 0 n.d. n.d. n.d. n.d. 0.01 0n.d. n.d. n.d. n.d. Human O —  4+ n.d. 1+ 2/3+ 4+ cells Incubated — 0n.d. 0  0 n.d. control Bench — 0 n.d. n.d. n.d. n.d. control

Example 10—Insertion of H_(di)-Sp-Ad-DOPE (VIII) and Galβ-Sp-Ad-DOPE(IX) Synthetic Glycolipids into Murine Red Blood Cells

The water soluble synthetic glycolipids designated H_(di)-sp-Ad-DOPE(VIII) and Galβ-sp-Ad-DOPE (IX) were prepared according to the methoddescribed in Example 1 with necessary modifications.

Murine RBCs were washed 3× in 1× PBS. 30 μl of packed RBCs were combinedwith 30 μl of H_(di)-sp-Ad-DOPE (VIII), and 30 μl of packed RBCs werecombined with 30 μl Galβ-sp-Ad-DOPE (IX), respectively. Both syntheticmolecule constructs were at a concentration of 1.0 mg/ml. 30 μl of 1×PBS was added to 30 μl of packed RBCs to act as the control group. Cellswere incubated for 90 minutes in a 37° C. shaking water-bath. RBCs werewashed 3× in 1× PBS.

Three groups of packed RBCs were incubated with an equal volume oflectin UEA-1 for 30 minutes at room temperature. The lectin was preparedin 1× PBS at a concentration of 0.1 mg/ml. 50 μl of a 3% cell suspensionwas spun for 15 seconds in an Immunofuge at low speed. Results were readby tube serology. The results are presented in Table 48. The resultsshow that neither anti-H IgM nor UEA-1 detects two sugars (H_(di)).

TABLE 47 Antisera used for results presented in Table 48. AntiseraManufacturer Batch Biotest anti-H Biotest AG UEA EY Labs 201105-2

TABLE 48 Murine RBCs transformed with Galβ-sp-Ad-DOPE or Hdi-sp-Ad-DOPE,assessed by agglutination. Cell Type Inserted Molecule UEA-1 MouseIgM^(H) Murine RBC Galβ (1 mg/ml) 0 n.d. Murine RBC H_(di) (1 mg/ml) 0 0Murine RBC Control (PBS) 0 0 Human RBC Control (PBS) 4+ 3+Abbreviations: n.d. Not determined

Example 11—Preparation of Sensitivity Controls

The synthetic glycolipids of the invention may be used in thepreparation of “sensitivity controls” (also referred to as “qualitycontrol cells”, “serology controls”, or “process controls”) as describedin the specification accompanying international application no.PCT/NZ02/00214 (WO 03/034074). The synthetic glycolipids provide theadvantage that the transformation of the RBCs may be achieved at reducedtemperatures.

RBC Transformation Solutions

Two stock solutions are used:

-   -   Solution 1: 1 mg/mL A_(tri)-sp-Ad-DOPE (I) suspended in        Celpresol™ solution.    -   Solution 2: 5 mg/mL B_(tri)-sp-Ad-DOPE (VI) suspended in        Celpresol™ solution.

Glycolipids are manufactured in a white dry powder. Glycolipids in thisform (enclosed in a sealed container under a controlled temperature) arestable for an indefinite period of time. The glycolipids are suspendedin solution (e.g. Celpresol™) by weight in order to formulate thetransformation solutions.

Once the transformation solutions are received at CSL, they are filtered(through a MILLEX®-GV 0.22 μfilter unit) under aseptic conditions.

Processing of RBCs

RBC donations are processed using a continuous flow centrifuge washerunder aseptic conditions. RBC donations are washed in buffered salinefollowed by Celpresol™ solution. The PCV of the RBC donations ismeasured on a Beckman Coulter AcT Diff analyser. The donations are thenadjusted to a packed cell volume (PCV) of 50% with the addition ofCelpresol™.

Transformation of RBCs to Provide “Weak AB Cells”

RBCs are washed in buffered saline and Celpresol™. The cells aresuspended in Celpresol™ solution to a PCV of >50%. The PCV of red cellsis measured using a Beckman Coulter AcT Diff. The mass of the red cellsolution is weighed.

The amount of A_(tri)-sp-Ad-DOPE (I), B_(tri)-sp-Ad-DOPE (VI) andCelpresol™ for transformation is calculated using the followingequations:

$a = \frac{P \times F}{S}$ $b = \frac{P \times F}{S}$c = P − (1 − P) − a − bwhere

-   -   a=amount of A_(tri)-sp-Ad-DOPE (I) to be added per 1 mL of red        cells (mL)    -   b=amount of B_(tri)-sp-Ad-DOPE (VI) to be added per 1 mL of red        cells (mL)    -   c=amount of Celpresol™ to be added per 1 mL of red cells (mL) to        dilute cells to 50% PCV    -   P=PCV of red cell solution    -   F=Final desired concentration of glycolipid    -   S=Concentration of stock glycolipid solution

To determine the amount of glycolipid and Celpresol™ to add to a bulksample of red cells, multiply each of a, b and c by the red cell volume.Add A_(tri)-sp-Ad-DOPE (I), B_(tri)-sp-Ad-DOPE (VI) and Celpresol™ tothe red cell bulk sample aseptically.

Incubate the sample for 3 hours at 20° C. under controlled temperatureconditions and constant gentle agitation. At the end of the 3 hourperiod, aseptically remove a sample of red cells and test the sample toconfirm transformation of the RBCs. Perform blood grouping using tube,tile and column agglutination technology (CAT) techniques.

Incubate the red cell sample for 3 hours at 2-8° C. under controlledtemperature conditions and constant gentle agitation for 18 hours. Atthe end of the 3 hour period, aseptically remove a sample of red cellsand test the sample to confirm transformation of the red cells. Performblood grouping using tube, tile and CAT techniques.

Wash the transformed red cells using a continuous flow centrifugemethod, under aseptic conditions using Celpresol™ solution. Measure thePCV of the washed red cells and adjust to 50% PCV by the addition ofCelpresol™ solution.

Formulation and Dispensing

Aseptically combine a volume of the transformed RBCs with a volume ofsimulated plasma diluent (SPD). The plasma may contain monoclonal andpolyclonal antibodies. Antibodies are selected according to the desiredcharacteristics of the sensitivity controls. The plasma may additionallycontain tartrazine and bovine serum albumin.

Blood grouping and antibody screening is performed on the bulk samplesusing tube, tile and CAT techniques. The transformed RBC-SPD blend isthen aseptically dispensed into BD Vacutainer tubes and the tubeslabelled accordingly.

Validation Testing

Weak AB cells produced by the use of synthetic glycolipids (designatedA_(w)B_(w) in Tables 51 to 53) were used to validate a range of testingplatforms in parallel with naturally occurring weak A, weak B and weakAB cells.

TABLE 49 Reagents and cards used in validation testing. Method ReagentTube Epiclone Tile Epiclone Ref Manufacturer and type Batch Expiry CAT 1OCD BioVue ABD/Rev ABR528A 16.06.05 CAT 2 OCD BioVue ABD/Rev ABR521A06.05.06 CAT 3 OCD BioVue ABD/ABD ACC255A 24.05.05 CAT 4 Diamed ID-MTS50092.10.02 Apr-05 CAT 5 Diamed ID-MTS Donor typing 51051.05.04 Mar-05CAT 6 Diamed ID-MTS Recipient typing 50053.07.02 Apr-05 CAT 7 DiamedID-MTS Cord typing 50961.08.03 Jul-05

TABLE 50 Testing platform methodology for validation testing. Tile 1drop 3% cells, 2 drops reagent, 15 min @ RT in moist chamber. Tube 2drops @ RT, 10 min. ID-MTS As per manufacturers instructions usingDil-2. BioVue As per manufacturers instructions using 0.8% RCD.

TABLE 51 Validation results across all methods against anti-A. Testingplatform Cell Type Tube Tile CAT 1 CAT 2 CAT 3 CAT 4 CAT 5 CAT 6 CAT 7 1A_(x) w+ 0 2+ 1+ 0 0 0 0 2 A_(x) w+ 0 2+ 2+ 0 0 0 0 3 A₁B 4+  4+ 4+  4+4+  4+  4+  4+  4+ 4 A_(x) w+ 0 2+ 2+ 0 0 0 0 5 A₂B 3+  3+ 4+ 3+  3+  1+ 2+  3+ 6 A_(x) w+ 0 2+ 2+ 0 0 0 0 7 A_(x) 1+ 0 2+ 2+ 0 0 0 0 8 A_(x) w+0 2+ 2+ 0 0 0 0 9 A_(x) 0  0 1+ 1+ 0 0 0 0 10 A_(x) w+ 0 2+ 2+ 0 0 0 011 A₃ 4+  4+ 4+ 3+  3+  1+  1+  3+ 12 A₃B 3+  3+ 3+ 3+  2+  w+  w+  2+13 B₃ 0  0 0  0 0  0 0 0 0 14 B₃ 0  0 0  0 0  0 0 0 0 15 A_(w)B_(w) 2+ 2+ 2+  2+ 2+ 0 0 0 0

TABLE 52 Validation results across all methods against anti-B. Testingplatform Cell Type Tube Tile CAT 1 CAT 2 CAT 3 CAT 4 CAT 5 CAT 6 CAT 7 1A_(x) 0 0 0 0 0 0 0 0 2 A_(x) 0 0 0 0 0 0 0 0 3 A₁B  4+  4+  4+ 4+  4+ 4+  3+  3+  4+ 4 A_(x) 0 0 0 0 0 0 0 0 5 A₂B  4+  4+  4+  4+  4+  3+ 3+  4+ 6 A_(x) 0 0 0 0 0 0 0 0 7 A_(x) 0 0 0 0 0 0 0 0 8 A_(x) 0 0 0 00 0 0 0 9 A_(x) 0 0 0 0 0 0 0 0 10 A_(x) 0 0 0 0 0 0 0 0 11 A₃ 0 0 0 0 00 0 0 12 A₃B  4+  4+  4+  4+  4+  4+  4+  4+ 13 B₃  2+  2+  3+ 2+  2+ 2+  2+  2+  2+ 14 B₃  2+  2+  2+ 2+  2+  2+  1+  1+  2+ 15 A_(w)B_(w) 3+  3+  1+ 1+  1+ 0 0 0 0

TABLE 53 Validation results across all methods against anti-AB. Testingplatform Cell Type Tube Tile CAT 1 CAT 2 CAT 3 CAT 4 CAT 5 CAT 6 CAT 7 1A_(x) 3+ 2+ 2+ 2 A_(x) 4+ 2+ 3+ 3 A₁B 4+ 4+ 4+ 4 A_(x) 3+ 2+ 3+ 5 A₂B 4+4+ 4+ 6 A_(x) 4+ 4+ 3+ 7 A_(x) 4+ 4+ 3+ 8 A_(x) 3+ 4+ 3+ 9 A_(x) 4+ 2+2+ 10 A_(x) 3+ 4+ 3+ 11 A₃ 4+ 4+ 4+ 12 A₃B 4+ 4+ 4+ 13 B₃ 2+ 2+ 2+ 14 B₃2+ 2+ 2+ 15 A_(w)B_(w) 3+ 3+ 3+

Example 12—Attachment of Modified Embryos to Transformed EndometrialCells

The ability to effect qualitative and quantitative differences in thecell surface antigens expressed by cell types other than RBCs wasinvestigated. The ability to enhance the adhesion of embryos toendometrial cells was adopted as a model system.

The synthetic molecules may be used as synthetic membrane anchors and/orsynthetic molecule constructs. Therefore, they may also be employed inthe method of enhancing embryo implantation as described ininternational patent application no PCT/NZ2003/000059 (published as WO03/087346) which is incorporated by reference.

Endometrial Cell Transformation

Insertion of Water Soluble Synthetic Molecule Construct A single cellsuspension of endometrial epithelial cells was prepared. The endometrialcells were washed 3× by resuspending in CMF HBSS and centrifuging at2000 rpm for 3 minutes. The washed cell preparation was resuspended in50 μl of M2.

Micro-centrifuge tubes each containing a 50 μl solution of 5M/mlendometrial cells were prepared. To separate tubes of endometrial cells50 μl of synthetic glycolipids A_(tri)-sp-Ad-DOPE (I) orB_(tri)-sp-Ad-DOPE A (VI), or 50 μl M2 were added to the control cells.The cells were incubated for 90 minutes at 37° C. on a mixer. Theendometrial cells were washed 3× by resuspending in CMF HBSS media andcentrifuging at 2000 rpm for 3 minutes. The washed cell preparation wasresuspended in 50 μl of M2.

Test for Insertion Using Fluorescent Probe:

50 μl of corresponding primary murine monoclonal antibody was added toeach tube. Each tube was incubated at room temperature for 10 minutes.Cells were washed 3× in M2 media. 10 μl of mouse anti-IgG FITC was addedto each tube. Tubes were incubated at room temperature in darkconditions for 10 minutes. Endometrial cells were mounted on glassslides and viewed under a fluorescence microscope.

Test for Direct Agglutination:

5 μl of each group of cells was placed onto separate microscope slides.To each 5 μl drop of cells 5 μl of a corresponding antibody was added.The cells were gently mixed on the slide for 2 minutes. Agglutinationwas visualised under the microscope. The results are presented in Table55.

TABLE 54 Antisera used for results presented in Table 55. AntiseraManufacturer Bioclone anti-A Ortho Diagnostics 01102 D.O.M. 16.05.02Bioclone anti-B Ortho Diagnostics Developmental reagent

TABLE 55 Endometrial cells transformed with A_(tri)-sp-Ad-DOPE (I) orB_(tri)-sp-Ad-DOPE A (VI), as visualised using fluorescence.Fluorescence Agglutination score after reaction by Inserted 1°incubation with microscopic Cell Type Antigen antibody IgFITC Probevisualisation Endometrial A_(tri)-sp-Ad- Anti-A 4+ 4+ cells DOPE (I)Bioclone (1 mg/ml) Endometrial B_(tri)-sp-Ad- Anti-B 1+ 3+ cells DOPE(VI) Bioclone (1 mg/ml) Endometrial Control (M2 Anti-A 0 0 cells media)Bioclone

Embryo Modification

Insertion of Water Soluble Synthetic Molecule Construct:

The embryo zona pellucida was removed by treating embryos with 0.5%pronase in a 37° C. oven for 6 minutes or until all zonas were removed.Micro-drops were prepared by adding 5 μl of synthetic glycolipidA_(tri)-sp-Ad-DOPE (I) or B_(tri)-sp-Ad-DOPE (VI), at a concentration of1 mg/mL to a 45 μl drop of M2 media overlaid with mineral oil. Allembryo groups were incubated in the 50 μl micro-drops for 1 hour at 37°C. Embryos from experimental and control groups were washed 3× with M2media.

Test for Insertion:

Embryos from experimental and control groups were placed into amicro-drop of corresponding antibody and incubated for 30 min at 37° C.Embryos from experimental and control groups were washed 3× with M2media.

Embryos from all experimental and control groups were placed intomicro-drops of anti-mouse Ig FITC (1:50 dilution anti-mouse Ig FITC inM2) and incubated for 30 min at 37° C. Embryos from experimental andcontrol groups were washed 3× with M2 media. Embryos were mounted onmicroscope slides in a 5 μl drop of M2 and the drops overlaid with oil.

The slides were viewed under a fluorescence microscope. Results arepresented in Tables 56 and 57. The negative result for transformationwith B_(tri)-sp-Ad-DOPE (VI) is attributed to a lack of 1° antibodysensitivity.

TABLE 56 Embryos transformed with Atri-sp-Ad-DOPE (I) as visualisedusing fluorescence. Fluorescence Embryo score after Morphology CellInserted 1° incubation with 24 hr post Type Antigen antibody IgFITCProbe insertion Embryos A_(tri)-sp-Ad- Anti-A 2+/1+ Appeared DOPE (I)Bioclone viable Embryos Control Anti-A 0 Appeared Bioclone viable

TABLE 57 Embryos transformed with A_(tri)-sp-Ad-DOPE (I) orB_(tri)-sp-Ad- DOPE (VI), as visualised using fluorescence. FluorescenceEmbryo score after Morphology Cell Inserted 1° incubation with 24 hrpost Type Antigen antibody IgFITC Probe insertion Embryos A_(tri)-sp-Ad-Anti-A 2+ n.d. DOPE (I) Bioclone Embryos B_(tri)-sp-Ad- Anti-B 0 n.d.DOPE (VI) Bioclone Embryos Control Anti-A 0 n.d. (M2 media) BiocloneEnhanced Attachment Transformed Endometrial Cells to Modified Embryos

Modified embryos (BioG-Avidin-BioIgG^(B) and BioG-Avidin-BioIgM^(A))were prepared in accordance with the methods described in thespecification accompanying the international application no.PCT/NZ03/00059 (published as WO03/087346).

Two concave glass slides were prepared, one with two wells of syntheticglycolipid A_(tri)-sp-Ad-DOPE (I) inserted endometrial cells and theother with two wells of synthetic glycolipid B_(tri)-sp-Ad-DOPE (VI)inserted endometrial cells.

The two groups of embryos were transferred to each of the concave glassslides:

Slide 1 A_(tri)/IgG^(B) embryos

-   -   A_(tri)/IgM^(A) embryos

Slide 2 B_(tri)/IgG^(B) embryos

-   -   B_(tri)/IgM^(A) embryos

The embryos were surrounded with endometrial cells. The wells werecovered with mineral oil and incubated for 15 minutes at 37° C. Using awide bore handling pipette each group of embryos were carefullytransferred to a fresh drop of M2 media. The embryos were gently washed.The embryos were gently transferred into 2 μL of M2 media on a markedmicroscope slide. Each drop was overlaid with mineral oil

Under a central plane of focus on an Olympus microscope the number ofendometrial cells adhered to the embryos in each group was assessed. Thenumber of cells adhered to each embryo was recorded. Results arepresented in Table 58.

TABLE 58 Endometrial cells transformed with A_(tri)-sp-Ad-DOPE (I) orB_(tri)-sp-Ad-DOPE (VI), and embryos modified with BioG-Avidin-BioIgGBor BioG-Avidin-BioIgMA. Assessment by attachment of endometrial cells toembryos. Average number of endometrial Transformed Modified cellsattached Cell Type endometrial cells embryos to modified embryosEndometrial A_(tri)-sp-Ad-DOPE BioG- 2.3 cells (I) Avidin- BioIgG^(B)BioG- 7.25 Avidin- BioIgM^(A) Endometrial B_(tri)-sp-Ad-DOPE BioG- 6.7cells (VI) Avidin- BioIgG^(B) BioG- 3.4 Avidin- BioIgM^(A)

Example 13

Association of KODE-Fluorescein (I) with Cell Membranes

KODE-fluorescein (I) readily associates with the membrane of red bloodcells.

Insertion of the molecule is observed when dispersions of the moleculeat concentrations greater than 0.1 mg/ml are contacted with suspensionsof the red blood cells.

A medium to strongly fluorescing cell was considered to indicate auniform distribution of the molecule across the cell membrane (FIG. 20).The incorporation and distribution appears to be stable for a period ofat least 40 days when cells are stored in the dark.

Where in the foregoing description reference has been made to integersor components having known equivalents then such equivalents are hereinincorporated as if individually set forth.

Although the invention has been described by way of example and withreference to possible embodiments thereof it is to be appreciated thatimprovements and/or modification may be made thereto without departingfrom the scope or spirit of the invention.

REFERENCES

-   Abe K, McKibbin J M & Hakomori S I. (1983) The monoclcnal antibody    directed to difucosylated type 2 chain    (Fucα1→42Galβ1→4[Fucα1→3]GlcNAc; Y determinant). J. Biol. Chem. 258:    11793-11797.-   Adamany A M, Blumenfeld O O, Sabo B & McCreary J. (1983) A    carbohydrate structural variant of MM glycoprotein (glycophorin    A). J. Biol. Chem. 258: 11537-11545.-   Blanchard D, Cartron J P, Fournet B, Mountreuil J, van Halbeek H &    Vliegenthart J F G. (1983) Primary structure of the oligosaccharide    determinant of blood group Cad specificity. J. Biol. Chem. 258:    7691-7695.-   Bovin, N. (2002) Neoglycoconjugates: Trade and art. Biochem. Soc.    Symp., 69, 143-160-   Fukuda M, Dell A & Fukuda M. (1984a) Structure of fetal    lactosaminoglycan. The carbohydrate moiety of band 3 isolated from    human umbilical cord erythrocytes. J. Biol. Chem. 259: 4782-4791.-   Fukuda M, Dell A, Oates J E & Fukuda M. (1984b) Structure of    branched lactosaminoglycan, the carbohydrate moiety of band 3    isolated from adult human erythrocytes. J. Biol. Chem. 259:    8260-8273.-   Fukuda M, Lauffenberger M, Sasaki H, Rogers M E & Dell A. (1987)    Structures of novel sialylated O-linked oligosaccharides isolated    from human erythrocyte glycophorins. J. Biol. Chem. 262:    11952-11957.-   Fukuda M N, Dell A, Oates J E, Wu P, Klock J C & Fukuda M. (1985)    Structures of glycosphingolipids isolated from human granulocytes.    The presence of a series of linear poly-N-acetyllactosaminylceramide    and its significance in glycolipids of whole blood cells. J. Biol.    Chem. 260: 1067-1082.-   Gillard B K, Blanchard D, Bouhours J F, Cartron J P, van Kuik J A,    Kamerling J P, Vliegenthart J F G & Marcus D M. (1988) Structure of    a ganglioside with Cad blood group antigen activity. Biochemistry.    27: 4601-4604.-   Hakomori S I, Nudelman E, Levery S B & Kannagi R. (1984) Novel    fucolipids accumulating in human adenocarcinoma. I. Glycilipids with    di- or trifucosylated type 2 chain. J. Biol. Chem. 259: 4672-4680-   Hanfland P, Kordowicz M, Niermann H, Egge H, Dabrowski U,    Peter-Katalinic J & Dabrowski J. (1984) Purification and structures    of branched blood-group-B-active glycosphingolipids from human    erythrocyte membranes. Eur. J. Biochem. 145: 531-542.-   Hanfland P, Kordowicz M, Peter-Katalinic J, Pfannschmidt G, Crawford    R J, Graham H A & Egge H. (1986) Immunochemistry of the Lewis    blood-group system: isolation and structures of Lewis-c active and    related glycosphingolipids from the plasma of blood-group O Le(a-b-)    nonsecretors. Arch. Biochem. Biophys. 246: 655-672.-   Hanfland P. (1975) Characterisation of B and H blood group active    glycosphingolipids from human B erythrocyte membranes. Chem. Phys.    Lipids. 15: 105-124.-   Hiraiwa N, Tsuyuoka K, Li Y T, Tanaka M, Seno T, Okubo Y, Fukuda Y,    Imura H & Kannagi R. (1990) Gangliosides and sialoglycoproteins    carrying a rare blood group antigen determinant, Cad, associated    with human cancers as detected by specific monoclonal antibodies.    Cancer Res. 50: 5497-5503.-   Kannagi R, Nudelman E, Levery S B, & Hakomori S I. (1982) A series    of human erythrocytes glycosphingolipids reacting to the monoclonal    antibody directed to a developmentally regulated antigen, SSEA-1. J.    Biol. Chem. 257: 14865-14874.-   Kewitz S, Groβ H J, Kosa R & Roelcke D. (1995) Anti=Pr cold    agglutinins recognise immunodominant α2,3- or α2,6-sialyl groups on    glycophorins. Glycocon. J. 12: 714-720.-   Koscielak J, Miller-Podraza H, Krauze R & Piasek A. (1976) Isolation    and characterisation of poly(glycosyl)ceramides (megaloglycolipids)    with A, H, and I blood group activities. Eur. J. Biochem. 71: 9-18.-   Laine R A. (1994) Invited commentary. Glycobiol 4: 759-767-   Lidowska E, Duk M & Dahr W. (1980) Comparison of alkali-labile    oligosaccharide chains of M and N blood-group glycopeptides from    human erythrocyte membrane. Carbohydr. Res. 79: 103-113.-   Lloyd K O & Kabat E A. (1968) Immunochemical studies on blood    groups. XLI. Proposed structures for the carbohydrate portions of    blood group A, B, H, Lewis^(a), and Lewis^(b) substances. Proc.    Natl. Acad. Sci USA. 61: 1470-1477.-   Lundblad A. (1977) Urinary glycoproteins, glycopeptides, and    oligosaccharides. In: The Glycoconjugates Eds Horowitz M I &    Pigman W. Vol 1: 441-458.-   Magnani J L, Nilsson B, Brockhaus M, Zopf D, Steplewski Z, Koprowski    H & Ginsburg V. (1986) A monoclonal antibody-defined antigen    associated with gastrointestinal cancer is a ganglioside containing    sialylated lacto-N-fucopentaose II. J. Biol. Chem. 257: 14365-14369.-   Nudelman E, Fukushi Y, Levery S B, Higuchi T & Hakomori S I. (1986)    Novel fucolipids of human adenocarcinoma: disialoyl Le^(a) antigen    (III⁴FucIII⁶NeuAcIV³NeuAcLc₄) of human colonic adenocarcinoma and    the monoclonal antibody (FH7) defining this structure. J. Biol.    Chem. 261: 5487-5495.-   Slomiany A, Zdebska E & Slomiany B L. (1984) Structures of the    neutral oligosaccharides isolated from A-active human gastric    mucin. J. Biol. Chem. 259: 14743-14749.-   Takasaki S, Yamashita K & Kobata A. (1978) The sugar chain    structures of ABO blood group active glycoproteins obtained from    human erythrocyte membrane. J. Biol. Chem. 253: 6086-6091.-   Tanaka M, Dube V E & Anderson B. (1984) Structures of    oligosaccharides cleaved by base-borohydride from an I, H, and    Le^(a) active ovarian cyst glycoprotein. Biochim. Biophys. Acta.    798: 283-290.-   Thomas D B & Winzler R J. (1969) Structural studies on human    erythrocytes glycoprotein. Alkali-labile oligosaccharides. J. Biol.    Chem. 244: 5943-5946.-   Watkins W M. (1966) Blood group substances. Science. 152: 172-181.-   Yoshima H, Furthmayr H & Kobata A. (1980) Structures of the    asparagine-linked sugar chains of glycophorin A. J. Biol. Chem. 255:    9713-9718.

The invention claimed is:
 1. A construct of the structure F-S₁-S₂-Lwhere: F—S₁ is an aminoalkylglycoside comprising the terminal sugarsGalα1-3Galβ and S₁ is 2-aminoethyl, 3-aminopropyl, 4-aminobutyl or5-aminopentyl; S₂ is —CO(CH₂)₂CO—, —CO(CH₂)₃CO—, —CO(CH₂)₄CO— or—CO(CH₂)₅CO—; and L is phosphatidylethanolamine.
 2. The construct ofclaim 1 where the aminoalkylglycoside is an aminoalkyltrisaccharide. 3.The construct of claim 2 where S₁ is 3-aminopropyl.
 4. The construct ofclaim 3 where S₂ is —CO(CH₂)₄CO—.
 5. The construct of claim 4 where L isdioleoylphosphatidylethanolamine.